Administrative Appointments

  • Director of Graduate Program, Dept. Microbiology and Immunology, Stanford University School of Medicine (2002 - Present)
  • Member of the Committee on Graduate Studies, Stanford University (2001 - 2004)
  • Member of School of Medicine Awards Committee, Stanford University School of Medicine (2005 - Present)
  • Chair, Dept. of Microbiology & Immunology, Stanford University School of Medicine (2010 - 2017)

Honors & Awards

  • Predoctoral Fellowship, Studienstiftung des Deutschen Volkes (1979-1982)
  • Postdoctoral Fellowship, Deutsche Forschungsgemeinschaft (1982-1985)
  • Faculty Research Award, American Cancer Society (1992-1997)
  • Editor, Virology (2003-present)
  • The Sidney and Skippy Frank Prize, Institute for Immunity, Transplantation and Infection, Stanford University (2006)
  • Merit Award, National Institutes of Health (2009-2019)
  • Elected, Fellow of the American Association for the Advancement of Science (2010)
  • Cozzarelli Prize, Proceedings of the National Academy of Sciences (2011)
  • Elected, Fellow of the American Society of Microbiology (2011)
  • NIH Director's Transformative R01 (T-R01) Program Award, National Institutes of Health (2011-2016)
  • Investigator, Chan Zuckerberg BioHub (2017-present)
  • Elected, Member of the National Academy of Sciences (2020)

Professional Education

  • Ph.D., SUNY at Stony Brook, Molecular Virology (1982)
  • B.S., University of Konstanz, Molecular Genetics (1979)

Current Research and Scholarly Interests

Our laboratory has been been studying the mechanism by which a liver-specific microRNA, miR-122, regulates the amplification of the hepatitis C virus (HCV) genome in cultured cells. Specifically, we have found that miR-122 interacts with the 5' end of the viral RNA and is essential for viral replication. Consequently, sequestration of miR-122 by antisense-oligonucleotides results in rapid loss of viral RNA. We are currently examining the mechanism by which miR-122 helps HCV RNA replication and are searching for cellular targets of miR-122 and their regulation by miR-122. These lines of investigations will lead to new insights how these small noncoding RNAs regulate expression of cellular and viral mRNAs and may point to new venues for antiviral therapeutics against HCV.

In a second line of investigation, we are studying the unusual mechanism of translation initiation by internal ribosome entry in certain viral (i.e. HCV, picornavirsues and some insect viruses) and cellular mRNA molecules. In the conventional scanning mechanism of translation initiation, which operates on most mRNA molecules, 40S subunits are recruited at or near the 5' end of the mRNA. Subsequently, the 40S ribosomal subunits are predicted to scan the mRNA in a 5' to 3' direction until the first AUG codon is encountered as start site for protein synthesis. However, certain viral and cellular mRNAs, notably encoding proto-oncogenes and regulatory genes, contain long 5' noncoding regions with multiple AUG codons. Thus, the translation initiation rate in these mRNAs is predicted to be low according to the scanning model; alternatively, other translation initiation mechanisms may operate to ensure efficient translation. Indeed, some of such mRNAs with long leaders contain internal ribosome entry sites which can bind ribosomes directly. Much of our work has been focussing on the mechanism and prevalence of internal ribosome binding. Specifically, we are addressing the following questions: Which cellular and viral mRNAs can be translated by internal ribosome binding? What are the cellular gene products that mediate internal ribosome binding? Is internal initiation regulated in the cell? What is the molecular basis for designating a given AUG codon as start site codon?

2023-24 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • Virus-derived circular RNAs populate hepatitis C virus-infected cells. Proceedings of the National Academy of Sciences of the United States of America Cao, Q. M., Boonchuen, P., Chen, T., Lei, S., Somboonwiwat, K., Sarnow, P. 2024; 121 (7): e2313002121


    It is known that pre-mRNAs in eukaryotic cells can be processed to circular RNAs by a backsplicing mechanism. Circular RNAs have great stability and can sequester proteins or small RNAs to exert functions on cellular pathways. Because viruses often exploit host pathways, we explored whether the RNA genome of the cytoplasmic hepatitis C virus is processed to yield virus-derived circRNAs (vcircRNAs). Computational analyses of RNA-seq experiments predicted that the viral RNA genome is fragmented to generate hundreds of vcircRNAs. More than a dozen of them were experimentally verified by rolling-circle amplification. VcircRNAs that contained the viral internal ribosome entry site were found to be translated into proteins that displayed proviral functions. Furthermore, two highly abundant, nontranslated vcircRNAs were shown to enhance viral RNA abundance. These findings argue that novel vcircRNA molecules modulate viral amplification in cells infected by a cytoplasmic RNA virus.

    View details for DOI 10.1073/pnas.2313002121

    View details for PubMedID 38319965

  • Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site. PLoS pathogens Mata, M. n., Neben, S. n., Majzoub, K. n., Carette, J. n., Ramanathan, M. n., Khavari, P. A., Sarnow, P. n. 2019; 15 (5): e1007467


    Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester mir-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.

    View details for PubMedID 31075158

  • Precursor microRNA-122 inhibits synthesis of Insig1 isoform mRNA by modulating polyadenylation site usage RNA Norman, K. L., Chen, T., Zeiner, G., Sarnow, P. 2017; 23 (12): 1886–93


    The insulin-induced gene 1 protein (Insig1) inhibits the cholesterol biosynthesis pathway by retaining transcription factor SREBP in the endoplasmic reticulum, and by causing the degradation of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. Liver-specific microRNA miR-122, on the other hand, enhances cholesterol biosynthesis by an unknown mechanism. We have found that Insig1 mRNAs are generated by alternative cleavage and polyadenylation, resulting in specific isoform mRNA species. During high cholesterol abundance, the short 1.4-kb Insig1 mRNA was found to be preferentially translated to yield Insig1 protein. Precursor molecules of miR-122 down-regulated the translation of the 1.4-kb Insig1 isoform mRNA by interfering with the usage of the promoter-proximal cleavage-polyadenylation site that gives rise to the 1.4-kb Insig1 mRNA. These findings argue that precursor miR-122 molecules modulate polyadenylation site usage in Insig1 mRNAs, resulting in down-regulation of Insig1 protein abundance. Thus, precursor microRNAs may have hitherto undetected novel functions in nuclear gene expression.

    View details for PubMedID 28928276

    View details for PubMedCentralID PMC5689008

  • Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase NATURE Jha, S., Rollins, M. G., Fuchs, G., Procter, D. J., Hall, E. A., Cozzolino, K., Sarnow, P., Savas, J. N., Walsh, D. 2017; 546 (7660): 651-+


    Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

    View details for PubMedID 28636603

    View details for PubMedCentralID PMC5526112

  • Making the Mark: The Role of Adenosine Modifications in the Life Cycle of RNA Viruses. Cell host & microbe Gonzales-van Horn, S. R., Sarnow, P. 2017; 21 (6): 661-669


    Viral epitranscriptomics is a newly emerging field that has identified unique roles for RNA modifications in modulating life cycles of RNA viruses. Despite the observation of a handful of modified viral RNAs five decades ago, very little was known about how these modifications regulate viral life cycles, until recently. Here we review the pro- and anti-viral effects of methyl-6-adenosine in distinct viral life cycles, the role of 2' O-methyl modifications in RNA stability and innate immune sensing, and functions of adenosine to inosine modifications in retroviral life cycles. With roles for over 100 modifications in RNA still unknown, this is a rapidly emerging field that is destined to suggest novel antiviral therapies.

    View details for DOI 10.1016/j.chom.2017.05.008

    View details for PubMedID 28618265

    View details for PubMedCentralID PMC5555051

  • "Escape from Transcriptional Shutoff during Poliovirus Infection: NF-kappa B-Responsive Genes I kappa Ba and A20" (vol 85, pg 10101, 2011) JOURNAL OF VIROLOGY Doukas, T., Sarnow, P. 2017; 91 (9)

    View details for PubMedID 28408611

    View details for PubMedCentralID PMC5391445

  • A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature Kim, H. K., Fuchs, G. n., Wang, S. n., Wei, W. n., Zhang, Y. n., Park, H. n., Roy-Chaudhuri, B. n., Li, P. n., Xu, J. n., Chu, K. n., Zhang, F. n., Chua, M. S., So, S. n., Zhang, Q. C., Sarnow, P. n., Kay, M. A. 2017; 552 (7683): 57–62


    Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

    View details for PubMedID 29186115

  • Unraveling the Mysterious Interactions Between Hepatitis C Virus RNA and Liver-Specific MicroRNA-122. Annual review of virology Sarnow, P., Sagan, S. M. 2016; 3 (1): 309-332


    Many viruses encode or subvert cellular microRNAs (miRNAs) to aid in their gene expression, amplification strategies, or pathogenic signatures. miRNAs typically downregulate gene expression by binding to the 3' untranslated region of their mRNA targets. As a result, target mRNAs are translationally repressed and subsequently deadenylated and degraded. Curiously, hepatitis C virus (HCV), a member of the Flaviviridae family, recruits two molecules of liver-specific microRNA-122 (miR-122) to the 5' end of its genome. In contrast to the canonical activity of miRNAs, the interactions of miR-122 with the viral genome promote viral RNA accumulation in cultured cells and in animal models of HCV infection. Sequestration of miR-122 results in loss of viral RNA both in cell culture and in the livers of chronic HCV-infected patients. This review discusses the mechanisms by which miR-122 is thought to enhance viral RNA abundance and the consequences of miR-122-HCV interactions. We also describe preliminary findings from phase II clinical trials in patients treated with miR-122 antisense oligonucleotides.

    View details for PubMedID 27578438

  • CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication. Viruses Shivaprasad, S., Qiao, W., Weng, K. F., Umashankar, P., Carette, J. E., Sarnow, P. 2024; 16 (5)


    The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

    View details for DOI 10.3390/v16050725

    View details for PubMedID 38793607

  • Variant enterovirus A71 found in immune-suppressed patient binds to heparan sulfate and exhibits neurotropism in B-cell-depleted mice. Cell reports Weng, K., Tee, H. K., Tseligka, E. D., Cagno, V., Mathez, G., Rosset, S., Nagamine, C. M., Sarnow, P., Kirkegaard, K., Tapparel, C. 2023; 42 (4): 112389


    Enterovirus A71 (EV-A71) causes hand, foot, and mouth disease outbreaks with neurological complications and deaths. We previously isolated an EV-A71 variant in the stool, cerebrospinal fluid, and blood of an immunocompromised patient who had a leucine-to-arginine substitution on the VP1 capsid protein, resulting in increased heparin sulfate binding. We show here that this mutation increases the virus's pathogenicity in orally infected mice with depleted B cells, which mimics the patient's immune status, and increases susceptibility to neutralizing antibodies. However, a double mutant with even greater heparin sulfate affinity is not pathogenic, suggesting that increased heparin sulfate affinity may trap virions in peripheral tissues and reduce neurovirulence. This research sheds light on the increased pathogenicity of variant with heparin sulfate (HS)-binding ability in individuals with decreased B cell immunity.

    View details for DOI 10.1016/j.celrep.2023.112389

    View details for PubMedID 37058406

  • Subversion of a protein-microRNA signaling pathway by hepatitis C virus. Proceedings of the National Academy of Sciences of the United States of America Cao, Q. M., Sarnow, P. 2023; 120 (4): e2220406120

    View details for DOI 10.1073/pnas.2220406120

    View details for PubMedID 36649406

  • Loquacious modulates flaviviral RNA replication in mosquito cells. PLoS pathogens Shivaprasad, S., Weng, K. F., Ooi, Y. S., Belk, J., Carette, J. E., Flynn, R., Sarnow, P. 2022; 18 (4): e1010163


    Arthropod-borne viruses infect both mosquito and mammalian hosts. While much is known about virus-host interactions that modulate viral gene expression in their mammalian host, much less is known about the interactions that involve inhibition, subversion or avoidance strategies in the mosquito host. A novel RNA-Protein interaction detection assay was used to detect proteins that directly or indirectly bind to dengue viral genomes in infected mosquito cells. Membrane-associated mosquito proteins Sec61A1 and Loquacious (Loqs) were found to be in complex with the viral RNA. Depletion analysis demonstrated that both Sec61A1 and Loqs have pro-viral functions in the dengue viral infectious cycle. Co-localization and pull-down assays showed that Loqs interacts with viral protein NS3 and both full-length and subgenomic viral RNAs. While Loqs coats the entire positive-stranded viral RNA, it binds selectively to the 3' end of the negative-strand of the viral genome. In-depth analyses showed that the absence of Loqs did not affect translation or turnover of the viral RNA but modulated viral replication. Loqs also displayed pro-viral functions for several flaviviruses in infected mosquito cells, suggesting a conserved role for Loqs in flavivirus-infected mosquito cells.

    View details for DOI 10.1371/journal.ppat.1010163

    View details for PubMedID 35482886

  • Cross-species microRNA transmission modulates flavivirus growth in mosquitoes. Trends in parasitology Shivaprasad, S., Sarnow, P. 2022


    Mosquitoes can be infected with a variety of RNA viruses. Recently,Zhu et al. demonstrated that human microRNA hsa-miR-150-5p is acquired by mosquitoes during blood meals and protects the Dengue virus by downregulation of chymotrypsin AaCT-1 mRNA. This finding suggests the use of microRNA antagomirs as an antiviral approach in mosquitoes.

    View details for DOI 10.1016/

    View details for PubMedID 35246384

  • An evolutionarily acquired microRNA shapes development of mammalian cortical projections. Proceedings of the National Academy of Sciences of the United States of America Diaz, J. L., Siththanandan, V. B., Lu, V., Gonzalez-Nava, N., Pasquina, L., MacDonald, J. L., Woodworth, M. B., Ozkan, A., Nair, R., He, Z., Sahni, V., Sarnow, P., Palmer, T. D., Macklis, J. D., Tharin, S. 2020


    The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are coexpressed across multiple projection neuron subtypes. Here, we discover 17 CSMN-enriched microRNAs (miRNAs), 15 of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is a demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians' increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.

    View details for DOI 10.1073/pnas.2006700117

    View details for PubMedID 33139574

  • The tale of two flaviviruses: subversion of host pathways by RNA shapes in dengue and hepatitis C viral RNA genomes. Current opinion in microbiology Shivaprasad, S., Sarnow, P. 2020; 59: 79–85


    Pathogenic RNA viruses continue to emerge owing to their rapid evolutionary rates. The family of the Flaviviridae contains enveloped, single-stranded, positive-sense RNA viruses that include mosquito borne viruses such as dengue virus and the blood-borne hepatitis C virus. Upon infection, the genomic viral RNA needs to first compete with a sea of host mRNAs for host ribosomes that synthesize the viral proteins. Then, the positive-sense template needs to be amplified and packaged into newly assembled virions. To accomplish these tasks, the virus subverts several biochemical machineries from the host. The participation of specific structures in the viral RNA mediates specific RNA-RNA and RNA-protein interactions that dictate many viral subversion strategies. In this review, we shall focus on the various mechanisms by which RNA elements in the dengue virus and hepatitis C virus untranslated regions aid the viral infectious cycle and contribute to viral fitness.

    View details for DOI 10.1016/j.mib.2020.08.007

    View details for PubMedID 33070015

  • Host-derived circular RNAs display proviral activities in Hepatitis C virus-infected cells. PLoS pathogens Chen, T. C., Tallo-Parra, M. n., Cao, Q. M., Kadener, S. n., Böttcher, R. n., Pérez-Vilaró, G. n., Boonchuen, P. n., Somboonwiwat, K. n., Díez, J. n., Sarnow, P. n. 2020; 16 (8): e1008346


    Viruses subvert macromolecular pathways in infected host cells to aid in viral gene amplification or to counteract innate immune responses. Roles for host-encoded, noncoding RNAs, including microRNAs, have been found to provide pro- and anti-viral functions. Recently, circular RNAs (circRNAs), that are generated by a nuclear back-splicing mechanism of pre-mRNAs, have been implicated to have roles in DNA virus-infected cells. This study examines the circular RNA landscape in uninfected and hepatitis C virus (HCV)-infected liver cells. Results showed that the abundances of distinct classes of circRNAs were up-regulated or down-regulated in infected cells. Identified circRNAs displayed pro-viral effects. One particular up-regulated circRNA, circPSD3, displayed a very pronounced effect on viral RNA abundances in both hepatitis C virus- and Dengue virus-infected cells. Though circPSD3 has been shown to bind factor eIF4A3 that modulates the cellular nonsense-mediated decay (NMD) pathway, circPSD3 regulates RNA amplification in a pro-viral manner at a post-translational step, while eIF4A3 exhibits the anti-viral property of the NMD pathway. Findings from the global analyses of the circular RNA landscape argue that pro-, and likely, anti-viral functions are executed by circRNAs that modulate viral gene expression as well as host pathways. Because of their long half-lives, circRNAs likely play hitherto unknown, important roles in viral pathogenesis.

    View details for DOI 10.1371/journal.ppat.1008346

    View details for PubMedID 32764824

  • An RNA-centric dissection of host complexes controlling flavivirus infection. Nature microbiology Ooi, Y. S., Majzoub, K., Flynn, R. A., Mata, M. A., Diep, J., Li, J. K., van Buuren, N., Rumachik, N., Johnson, A. G., Puschnik, A. S., Marceau, C. D., Mlera, L., Grabowski, J. M., Kirkegaard, K., Bloom, M. E., Sarnow, P., Bertozzi, C. R., Carette, J. E. 2019


    Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.

    View details for DOI 10.1038/s41564-019-0518-2

    View details for PubMedID 31384002

  • Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site PLOS PATHOGENS Mata, M., Neben, S., Majzoub, K., Carette, J., Ramanathan, M., Khavari, P. A., Sarnow, P. 2019; 15 (5)
  • Enterovirus pathogenesis requires the host methyltransferase SETD3. Nature microbiology Diep, J. n., Ooi, Y. S., Wilkinson, A. W., Peters, C. E., Foy, E. n., Johnson, J. R., Zengel, J. n., Ding, S. n., Weng, K. F., Laufman, O. n., Jang, G. n., Xu, J. n., Young, T. n., Verschueren, E. n., Kobluk, K. J., Elias, J. E., Sarnow, P. n., Greenberg, H. B., Hüttenhain, R. n., Nagamine, C. M., Andino, R. n., Krogan, N. J., Gozani, O. n., Carette, J. E. 2019


    Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.

    View details for DOI 10.1038/s41564-019-0551-1

    View details for PubMedID 31527793

  • microRNAs Refine Cortical Projection Neuron Subtype during Mammalian Development Siththanandan, V., Diaz, J., Lu, V., Gonzalez-Nava, N., Pasquina, L., MacDonald, J., Woodworth, M., Sahni, V., Sarnow, P., Palmer, T., Macklis, J., Tharin, S. WILEY. 2018: S276–S277
  • A transfer RNA derived small RNA affects translation in rapidly dividing cells and a target for hepatocellular carcinoma Kim, H., Fuchs, G., Wang, S., Wei, W., Zhang, Y., Park, H., Roy-Chaudhuri, B., Li, P., Xu, J., Chu, K., Zhang, F., Chua, M., So, S., Zhang, C., Sarnow, P., Kay, M. A. AMER ASSOC CANCER RESEARCH. 2018
  • Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens NATURE Marceau, C. D., Puschnik, A. S., Majzoub, K., Ooi, Y. S., Brewer, S. M., Fuchs, G., Swaminathan, K., Mata, M. A., Elias, J. E., Sarnow, P., Carette, J. E. 2016; 535 (7610): 159-?


    The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

    View details for DOI 10.1038/nature18631

    View details for Web of Science ID 000379015600044

    View details for PubMedID 27383987

    View details for PubMedCentralID PMC4964798

  • cis-Acting RNA elements in the hepatitis C virus RNA genome VIRUS RESEARCH Sagan, S. M., Chahal, J., Sarnow, P. 2015; 206: 90-98


    Hepatitis C virus (HCV) infection is a rapidly increasing global health problem with an estimated 170 million people infected worldwide. HCV is a hepatotropic, positive-sense RNA virus of the family Flaviviridae. As a positive-sense RNA virus, the HCV genome itself must serve as a template for translation, replication and packaging. The viral RNA must therefore be a dynamic structure that is able to readily accommodate structural changes to expose different regions of the genome to viral and cellular proteins to carry out the HCV life cycle. The ∼9600 nucleotide viral genome contains a single long open reading frame flanked by 5' and 3' non-coding regions that contain cis-acting RNA elements important for viral translation, replication and stability. Additional cis-acting RNA elements have also been identified in the coding sequences as well as in the 3' end of the negative-strand replicative intermediate. Herein, we provide an overview of the importance of these cis-acting RNA elements in the HCV life cycle.

    View details for DOI 10.1016/j.virusres.2014.12.029

    View details for Web of Science ID 000357438700011

    View details for PubMedID 25576644

  • Supporting Role for GTPase Rab27a in Hepatitis C Virus RNA Replication through a Novel miR-122-Mediated Effect. PLoS pathogens Chen, T., Hsieh, C., Sarnow, P. 2015; 11 (8)


    The small GTPase Rab27a has been shown to control membrane trafficking and microvesicle transport pathways, in particular the secretion of exosomes. In the liver, high expression of Rab27a correlates with the development of hepatocellular carcinoma. We discovered that low abundance of Rab27a resulted in decreased hepatitis C virus (HCV) RNA and protein abundances in virus-infected cells. Curiously, both cell-associated and extracellular virus yield decreased in Rab27a depleted cells, suggesting that reduced exosome secretion did not cause the observed effect. Instead, Rab27a enhanced viral RNA replication by a mechanism that involves the liver-specific microRNA miR-122. Rab27a surrounded lipid droplets and was enriched in membrane fractions that harbor viral replication proteins, suggesting a supporting role for Rab27a in viral gene expression. Curiously, Rab27a depletion decreased the abundance of miR-122, whereas overexpression of miR-122 in Rab27a-depleted cells rescued HCV RNA abundance. Because intracellular HCV RNA abundance is enhanced by the binding of two miR-122 molecules to the extreme 5' end of the HCV RNA genome, the diminished amounts of miR-122 in Rab27a-depleted cells could have caused destabilization of HCV RNA. However, the abundance of HCV RNA carrying mutations on both miR-122-binding sites and whose stability was supported by ectopically expressed miR-122 mimetics with compensatory mutations also decreased in Rab27a-depleted cells. This result indicates that the effect of Rab27a depletion on HCV RNA abundance does not depend on the formation of 5' terminal HCV/miR-122 RNA complexes, but that miR-122 has a Rab27a-dependent function in the HCV lifecycle, likely the downregulation of a cellular inhibitor of HCV gene expression. These findings suggest that the absence of miR-122 results in a vulnerability not only to exoribonucleases that attack the viral genome, but also to upregulation of one more cellular factor that inhibit viral gene expression.

    View details for DOI 10.1371/journal.ppat.1005116

    View details for PubMedID 26305877

    View details for PubMedCentralID PMC4549268

  • Supporting Role for GTPase Rab27a in Hepatitis C Virus RNA Replication through a Novel miR-122-Mediated Effect PLOS PATHOGENS Chen, T., Hsieh, C., Sarnow, P. 2015; 11 (8)
  • Interaction of Host Cell microRNAs with the HCV RNA Genome during Infection of Liver Cells SEMINARS IN LIVER DISEASE Sedano, C. D., Sarnow, P. 2015; 35 (1): 75-80


    It has remained an enigma how hepatitis C viral (HCV) RNA can persist in the liver of infected patients for many decades. With the recent discovery of roles for microRNAs in gene expression, it was reported that the HCV RNA genome subverts liver-specific microRNA miR-122 to protect its 5' end from degradation by host cell exoribonucleases. Sequestration of miR-122 in cultured liver cells and in the liver of chimpanzees by small, modified antisense RNAs resulted in dramatic loss of HCV RNA and viral yield. This finding led to the first successful human trial in which subcutaneous administration of antisense molecules against miR-122 lowered viral yield in HCV patients, without the emergence of resistant virus. In this review, the authors summarize the molecular mechanism by which miR-122 protects the HCV RNA genome from degradation by exoribonucleases Xrn1 and Xrn2 and discuss the application of miR-122 antisense molecules in the clinic.

    View details for DOI 10.1055/s-0034-1397351

    View details for Web of Science ID 000348769600009

    View details for PubMedID 25632937

  • Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site. Proceedings of the National Academy of Sciences of the United States of America Fuchs, G., Petrov, A. N., Marceau, C. D., Popov, L. M., Chen, J., O'Leary, S. E., Wang, R., Carette, J. E., Sarnow, P., Puglisi, J. D. 2015; 112 (2): 319-325


    Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

    View details for DOI 10.1073/pnas.1421328111

    View details for PubMedID 25516984

  • Dissecting noncoding and pathogen RNA-protein interactomes RNA-A PUBLICATION OF THE RNA SOCIETY Flynn, R. A., Martin, L., Spitale, R. C., Do, B. T., Sagan, S. M., Zarnegar, B., Qu, K., Khavari, P. A., Quake, S. R., Sarnow, P., Chang, H. Y. 2015; 21 (1): 135-143


    RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

    View details for DOI 10.1261/rna.047803.114

    View details for PubMedID 25411354

  • Hepatitis C Virus Subverts Liver-Specific miR-122 to Protect the Viral Genome from Exoribonuclease Xrn2. Cell host & microbe Sedano, C. D., Sarnow, P. 2014; 16 (2): 257-264


    The abundant, liver-specific microRNA miR-122 forms extensive base-pairing interactions with the 5' noncoding region of the hepatitis C virus (HCV) RNA genome, protecting the viral RNA from degradation. We discovered that the 5'-3' exoribonuclease Xrn2, which plays a crucial role in the transcription termination of RNA polymerase II, modulates HCV RNA abundance in the cytoplasm, but is counteracted by miR-122-mediated protection. Specifically, Xrn2 depletion results in increased accumulation of viral RNA, while Xrn2 overexpression diminishes viral RNA abundance. Depletion of Xrn2 did not alter translation or replication rates of HCV RNA, but affected viral RNA stability. Importantly, during sequestration of miR-122, Xrn2 depletion restored HCV RNA abundance, arguing that Xrn2 depletion eliminates the miR-122 requirement for viral RNA stability. Thus, Xrn2 has a cytoplasmic, antiviral function against HCV that is counteracted by HCV's subversion of miR-122 to form a protective oligomeric complex at the 5' end of the viral genome.

    View details for DOI 10.1016/j.chom.2014.07.006

    View details for PubMedID 25121753

  • Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension. journal of experimental medicine Sawada, H., Saito, T., Nickel, N. P., Alastalo, T., Glotzbach, J. P., Chan, R., Haghighat, L., Fuchs, G., Januszyk, M., Cao, A., Lai, Y., Perez, V. d., Kim, Y., Wang, L., Chen, P., Spiekerkoetter, E., Mitani, Y., Gurtner, G. C., Sarnow, P., Rabinovitch, M. 2014; 211 (2): 263-280


    Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34-PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)-positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

    View details for DOI 10.1084/jem.20111741

    View details for PubMedID 24446489

  • Enhancement of hepatitis C viral RNA abundance by precursor miR-122 molecules RNA-A PUBLICATION OF THE RNA SOCIETY Cox, E. M., Sagan, S. M., Mortimer, S. A., Doudna, J. A., Sarnow, P. 2013; 19 (12): 1825-1832


    The hepatitis C viral RNA genome forms a complex with liver-specific microRNA (miR-122) at the extreme 5' end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. The abundances of primary and precursor forms of miR-122, but not the abundance of mature miR-122, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of precursor molecules of miR-122 in regulating viral gene expression. Modified precursor molecules of miR-122 were synthesized that were refractory to cleavage by Dicer. These molecules were found to enhance the abundance of HCV RNA. Furthermore, they diminished the expression of mRNAs that contained binding sites for miR-122 in their 3' noncoding regions. By use of duplex and precursor miR-122 mimetic molecules that carried mutations in the passenger strand of miR-122, the effects on viral and reporter gene expression could be pinpointed to the action of precursor miR-122 molecules. Targeting the circadian expression of precursor miR-122 by specific compounds likely provides novel therapeutic strategies.

    View details for DOI 10.1261/rna.040865.113

    View details for Web of Science ID 000327445700021

    View details for PubMedID 24106328

    View details for PubMedCentralID PMC3884667

  • Molecular biology. RNAi, Antiviral after all. Science Sagan, S. M., Sarnow, P. 2013; 342 (6155): 207-208

    View details for DOI 10.1126/science.1245475

    View details for PubMedID 24115433

  • Modulation of GB Virus B RNA Abundance by MicroRNA-122: Dependence on and Escape from MicroRNA-122 Restriction. Journal of virology Sagan, S. M., Sarnow, P., Wilson, J. A. 2013; 87 (13): 7338-7347


    Hepatitis C virus (HCV) RNA forms an unusual interaction with human microRNA-122 (miR-122) that promotes viral RNA accumulation in cultured human liver cells and in the livers of infected chimpanzees. GB virus B (GBV-B) is a hepatotropic virus and close relative of HCV. Thus, GBV-B has been used as a surrogate system to study HCV amplification in cultured cells and in infected tamarins. It was discovered that the 5' terminal sequences of GBV-B RNA, like HCV RNA, forms an Argonaute 2-mediated complex with two miR-122 molecules that are essential for accumulation of GBV-B subgenomic replicon RNA. However, sequences in miR-122 that anneal to each viral RNA genome were different, suggesting distinct overall structural features in HCV:miR-122 and GBV-B:miR-122 complexes. Surprisingly, a deletion that removed both miR-122 binding sites from the subgenomic GBV-B RNAs rendered viral RNA amplification independent from miR-122 and Argonaute 2. This finding suggests that structural features at the end of the viral genome dictate whether miR-122 is required to aid in maintaining viral RNA abundance.

    View details for DOI 10.1128/JVI.00378-13

    View details for PubMedID 23616647

  • Reduced DEAF1 function during type 1 diabetes inhibits translation in lymph node stromal cells by suppressing Eif4g3. Journal of molecular cell biology Yip, L., Creusot, R. J., Pager, C. T., Sarnow, P., Fathman, C. G. 2013; 5 (2): 99-110


    The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.

    View details for DOI 10.1093/jmcb/mjs052

    View details for PubMedID 22923498

    View details for PubMedCentralID PMC3604916

  • Reduced DEAF1 function during type 1 diabetes inhibits translation in lymph node stromal cells by suppressing Eif4g3 JOURNAL OF MOLECULAR CELL BIOLOGY Yip, L., Creusot, R. J., Pager, C. T., Sarnow, P., Fathman, C. G. 2013; 5 (2): 99-110


    The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.

    View details for DOI 10.1093/jmcb/mjs052

    View details for Web of Science ID 000316701200004

    View details for PubMedID 22923498

    View details for PubMedCentralID PMC3604916

  • Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules. Virology Pager, C. T., Schütz, S., Abraham, T. M., Luo, G., Sarnow, P. 2013; 435 (2): 472-484


    Components of cytoplasmic processing bodies (P-bodies) and stress granules can be subverted during viral infections to modulate viral gene expression. Because hepatitis C virus (HCV) RNA abundance is regulated by P-body components such as microRNA miR-122, Argonaute 2 and RNA helicase RCK/p54, we examined whether HCV infection modulates P-bodies and stress granules during viral infection. It was discovered that HCV infection decreased the number of P-bodies, but induced the formation of stress granules. Immunofluorescence studies revealed that a number of P-body and stress granule proteins co-localized with viral core protein at lipid droplets, the sites for viral RNA packaging. Depletion of selected P-body proteins decreased overall HCV RNA and virion abundance. Depletion of stress granule proteins also decreased overall HCV RNA abundance, but surprisingly enhanced the accumulation of infectious, extracellular virus. These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm.

    View details for DOI 10.1016/j.virol.2012.10.027

    View details for PubMedID 23141719

    View details for PubMedCentralID PMC3534916

  • Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules VIROLOGY Pager, C. T., Schuetz, S., Abraham, T. M., Luo, G., Sarnow, P. 2013; 435 (2): 472-484
  • Protection of the hepatitis C viral RNA genome and modulation of polyadenylation site usage in Insig1 mRNA by liver-specific pre- and mature microRNA 122 8th Annual Meeting of the Oligonucleotide-Therapeutics-Society Sarnow, P., Machlin, E., Sagan, S. MARY ANN LIEBERT INC. 2012: A8–A9
  • Combating Hepatitis C Virus by Targeting MicroRNA-122 Using Locked Nucleic Acids CURRENT GENE THERAPY Machlin, E. S., Sarnow, P., Sagan, S. M. 2012; 12 (4): 301-306


    MicroRNAs have been predicted to regulate the stability and translation of many target mRNAs that are involved in modulating disease outcome. Thus, valuable strategies to enhance or to diminish the function of microRNAs are needed to manipulate microRNA-mediated target gene expression. Recently, it has become apparent that one class of antisense oligonucleotides, locked nucleic acids, can be used to sequester microRNAs in the liver of a variety of animals including humans, opening the possibility of applying locked nucleic acid-mediated gene therapy. This review summarizes the success of sequestration of liver-specific microRNA miR-122 by antisense locked nucleic acids and their use in combating hepatitis C virus in clinical trials.

    View details for Web of Science ID 000306834700006

    View details for PubMedID 22856605

  • Reduced BMPR2 Increases GM-CSF mRNA Translation by Inhibiting eIF2 alpha Mediated Stress Granule Formation and Propensity to Pulmonary Vascular Disease Scientific Sessions of the American-Heart-Association/Resuscitation Science Symposium Sawada, H., Alastalo, T., Glotzbach, J. P., Chan, R., Fuchs, G., Januszyk, M., Lai, Y., Perez, V. D., Saito, T., Spiekerkoetter, E., Wang, L., Gurtner, G. C., Sarnow, P., Rabinovitch, M. LIPPINCOTT WILLIAMS & WILKINS. 2011
  • Escape from Transcriptional Shutoff during Poliovirus Infection: NF-kappa B-Responsive Genes I kappa Ba and A20 JOURNAL OF VIROLOGY Doukas, T., Sarnow, P. 2011; 85 (19): 10101-10108


    It has been known for a long time that infection of cultured cells with poliovirus results in the overall inhibition of transcription of most host genes. We examined whether selected host genes can escape transcriptional inhibition by thiouridine marking newly synthesized host mRNAs during viral infection. Using cDNA microarrays hybridized to cDNAs made from thiolated mRNAs, a small set of host transcripts was identified and their expression verified by quantitative PCR and Northern and Western blot analyses. These transcripts were synthesized from genes that displayed enrichment for NF-κB binding sites in their promoter regions, suggesting that some NF-κB-regulated promoters can escape the virus-induced inhibition of transcription. In particular, two negative regulators of NF-κB, IκBa and A20, were upregulated during viral infection. Depletion of A20 enhanced viral RNA abundance and viral yield, arguing that cells respond to virus infection by counteracting NF-κB-induced proviral effects.

    View details for DOI 10.1128/JVI.00575-11

    View details for Web of Science ID 000296253900042

    View details for PubMedID 21795344

    View details for PubMedCentralID PMC3196431

  • Proteomic Analysis of Ribosomes: Translational Control of mRNA Populations by Glycogen Synthase GYS1 JOURNAL OF MOLECULAR BIOLOGY Fuchs, G., Diges, C., Kohlstaedt, L. A., Wehner, K. A., Sarnow, P. 2011; 410 (1): 118-130


    Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins, and numerous associated "nonribosomal" proteins. To identify nonribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, glycogen synthase 1 (GYS1), was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, by virtue of its ribosomal association, provides a feedback loop between the energy state of the cells and the translation machinery.

    View details for DOI 10.1016/j.jmb.2011.04.064

    View details for Web of Science ID 000292440100011

    View details for PubMedID 21570405

    View details for PubMedCentralID PMC3131224

  • Masking the 5 ' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Machlin, E. S., Sarnow, P., Sagan, S. M. 2011; 108 (8): 3193-3198


    Hepatitis C virus subverts liver-specific microRNA, miR-122, to upregulate viral RNA abundance in both infected cultured cells and in the liver of infected chimpanzees. These findings have identified miR-122 as an attractive antiviral target. Thus, it is imperative to know whether a distinct functional complex exists between miR-122 and the viral RNA versus its normal cellular target mRNAs. Toward this goal, effects on viral RNA abundance of mutated miR-122 duplex molecules, bound at each of the two target sites in the viral genome, were compared to effects on microRNA- or siRNA-mediated regulation of reporter target mRNAs. It was found that miR-122 formed an unusual microRNA complex with the viral RNA that is distinct from miR-122 complexes with reporter mRNAs. Notably, miR-122 forms an oligomeric complex in which one miR-122 molecule binds to the 5' terminus of the hepatitis C virus (HCV) RNA with 3' overhanging nucleotides, masking the 5' terminal sequences of the HCV genome. Furthermore, specific internal nucleotides as well as the 3' terminal nucleotides in miR-122 were absolutely required for maintaining HCV RNA abundance but not for microRNA function. Both miR-122 molecules utilize similar internal nucleotides to interact with the viral genome, creating a bulge and tail in the miR-122 molecules, revealing tandemly oriented oligomeric RNA complexes. These findings suggest that miR-122 protects the 5' terminal viral sequences from nucleolytic degradation or from inducing innate immune responses to the RNA terminus. Finally, this remarkable microRNA-mRNA complex could be targeted with compounds that inactivate miR-122 or interfere with this unique RNA structure.

    View details for DOI 10.1073/pnas.1012464108

    View details for Web of Science ID 000287580400026

    View details for PubMedID 21220300

    View details for PubMedCentralID PMC3044371

  • RNA Virus Harnesses MicroRNAs to Seize Host Translation Control CELL HOST & MICROBE Abraham, T. M., Sarnow, P. 2011; 9 (1): 5-7


    Picornaviruses have evolved elaborate strategies to subvert host translation. In this issue of Cell Host and Microbe, Ho et al. (2011) report that enterovirus infection induces the synthesis of a transcription factor that enhances the synthesis of microRNA-141, which suppresses translation of the cap-binding protein, eIF4E, mRNA to inhibit cap-dependent translation.

    View details for DOI 10.1016/j.chom.2011.01.004

    View details for Web of Science ID 000287344500003

    View details for PubMedID 21238942

  • Herpes Simplex Virus is Akt-ing in translational control GENES & DEVELOPMENT Norman, K. L., Sarnow, P. 2010; 24 (23): 2583-2586


    All viruses depend on the cellular protein synthesis machinery for the production of viral proteins. Thus, viruses have evolved a variety of strategies to avoid innate host responses that inhibit protein synthesis. In this issue of Genes & Development, Chuluunbaatar and colleagues (pp. 2627-2639) demonstrate that Herpes Simplex Virus-1 counteracts this response through viral kinase Us3, which mimics cellular kinase Akt to phosphorylate and repress tuberous sclerosis complex 2 (TSC2), resulting in the activation of mammalian target of rapamycin complex 1 (mTORC1) and enhancement of mRNA translation.

    View details for DOI 10.1101/gad.2004510

    View details for Web of Science ID 000284835800001

    View details for PubMedID 21123644

    View details for PubMedCentralID PMC2994031

  • Hepatitis C Virus Core-Derived Peptides Inhibit Genotype 1b Viral Genome Replication via Interaction with DDX3X PLOS ONE Sun, C., Pager, C. T., Luo, G., Sarnow, P., Cate, J. H. 2010; 5 (9)


    The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in vivo. However, the specific interactions between these two proteins and the functional importance of these interactions for the HCV viral life cycle remain unclear. We show that amino acids 16-36 near the N-terminus of the HCV core protein interact specifically with DDX3X both in vitro and in vivo. Replication of HCV replicon NNeo/C-5B RNA (genotype 1b) is significantly suppressed in HuH-7-derived cells expressing green fluorescent protein (GFP) fusions to HCV core protein residues 16-36, but not by GFP fusions to core protein residues 16-35 or 16-34. Notably, the inhibition of HCV replication due to expression of the GFP fusion to HCV core protein residues 16-36 can be reversed by overexpression of DDX3X. These results suggest that the protein interface on DDX3X that binds the HCV core protein is important for replicon maintenance. However, infection of HuH-7 cells by HCV viruses of genotype 2a (JFH1) was not affected by expression of the GFP fusion protein. These results suggest that the role of DDX3X in HCV infection involves aspects of the viral life cycle that vary in importance between HCV genotypes.

    View details for DOI 10.1371/journal.pone.0012826

    View details for Web of Science ID 000281960800021

    View details for PubMedID 20862261

  • Plasmacytoid dendritic cells as guardians in hepatitis C virus-infected liver PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sagan, S. M., Sarnow, P. 2010; 107 (17): 7625-7626

    View details for DOI 10.1073/pnas.1002943107

    View details for Web of Science ID 000277088700007

    View details for PubMedID 20404186

    View details for PubMedCentralID PMC2867855

  • OGFOD1, a Novel Modulator of Eukaryotic Translation Initiation Factor 2 alpha Phosphorylation and the Cellular Response to Stress MOLECULAR AND CELLULAR BIOLOGY Wehner, K. A., Schuetz, S., Sarnow, P. 2010; 30 (8): 2006-2016


    Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2alpha and the eIF2alpha kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.

    View details for DOI 10.1128/MCB.01350-09

    View details for Web of Science ID 000275980900013

    View details for PubMedID 20154146

    View details for PubMedCentralID PMC2849474

  • Stable Formation of Compositionally Unique Stress Granules in Virus-Infected Cells JOURNAL OF VIROLOGY Piotrowska, J., Hansen, S. J., Park, N., Jamka, K., Sarnow, P., Gustin, K. E. 2010; 84 (7): 3654-3665


    Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.

    View details for DOI 10.1128/JVI.01320-09

    View details for Web of Science ID 000275307400049

    View details for PubMedID 20106928

  • Hepatitis C virus' Achilles' heel - dependence on liver-specific microRNA miR-122 CELL RESEARCH Norman, K. L., Sarnow, P. 2010; 20 (3): 247-249

    View details for DOI 10.1038/cr.2010.28

    View details for Web of Science ID 000275816300001

    View details for PubMedID 20190773

  • Temperature Protects Insect Cells from Infection by Cricket Paralysis Virus JOURNAL OF VIROLOGY Cevallos, R. C., Sarnow, P. 2010; 84 (3): 1652-1655


    Heat shock is a well-known stress response characterized by a rapid synthesis of a set of proteins which are responsible for protection against stress. We examined the role of temperature on the growth of cricket paralysis virus, a member of the family Dicistroviridae, in insect cells. Heat shock caused an induction of heat shock protein-encoding mRNAs in uninfected cells but not in infected cells. While viral RNA and protein were abundant during heat shock, virion formation was inhibited at higher temperatures. The different susceptibility to pathogens at different temperatures is likely a crucial feature of host-pathogen interaction in cold-blooded animals.

    View details for DOI 10.1128/JVI.01730-09

    View details for Web of Science ID 000273531600044

    View details for PubMedID 19906924

    View details for PubMedCentralID PMC2812312

  • Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems PLOS PATHOGENS Parameswaran, P., Sklan, E., Wilkins, C., Burgon, T., Samuel, M. A., Lu, R., Ansel, K. M., Heissmeyer, V., Einav, S., Jackson, W., Doukas, T., Paranjape, S., Polacek, C., dos Santos, F. B., Jalili, R., Babrzadeh, F., Gharizadeh, B., Grimm, D., Kay, M., Koike, S., Sarnow, P., Ronaghi, M., Ding, S., Harris, E., Chow, M., Diamond, M. S., Kirkegaard, K., Glenn, J. S., Fire, A. Z. 2010; 6 (2)


    We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

    View details for DOI 10.1371/journal.ppat.1000764

    View details for PubMedID 20169186

  • Modulation of Hepatitis C Virus RNA Abundance and the Isoprenoid Biosynthesis Pathway by MicroRNA miR-122 Involves Distinct Mechanisms JOURNAL OF VIROLOGY Norman, K. L., Sarnow, P. 2010; 84 (1): 666-670


    MicroRNA 122 (miR-122) promotes hepatitis C virus (HCV) RNA abundance through a direct interaction with the viral RNA and stimulates the mevalonate pathway in the animal liver. We found that overexpression of miR-122 enhanced viral RNA accumulation without affecting genes in the mevalonate pathway, such as the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) gene. However, inhibition of miR-122 decreased both HCV RNA and HMGCR RNA with little effects on the rates of HCV and HMGCR RNA synthesis. Loss of HCV RNA could not be restored by isoprenoid intermediate metabolites. Overall, these findings suggest that miR-122 modulates viral RNA abundance independently of its effect on isoprenoid metabolism.

    View details for DOI 10.1128/JVI.01156-09

    View details for Web of Science ID 000272564300063

    View details for PubMedID 19846523

    View details for PubMedCentralID PMC2798415

  • The Imd Pathway Is Involved in Antiviral Immune Responses in Drosophila PLOS ONE Costa, A., Jan, E., Sarnow, P., Schneider, D. 2009; 4 (10)


    Cricket Paralysis virus (CrPV) is a member of the Dicistroviridae family of RNA viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway.

    View details for DOI 10.1371/journal.pone.0007436

    View details for Web of Science ID 000270847800002

    View details for PubMedID 19829691

    View details for PubMedCentralID PMC2758544

  • LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor JOURNAL OF CELL SCIENCE Ying, L., Lau, A., Alvira, C. M., West, R., Cann, G. M., Zhou, B., Kinnear, C., Jan, E., Sarnow, P., van de Rijn, M., Rabinovitch, M. 2009; 122 (9): 1441-1451


    Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3' UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types.

    View details for DOI 10.1242/jcs.025957

    View details for PubMedID 19366727

  • Biological basis for restriction of microRNA targets to the 3 ' untranslated region in mammalian mRNAs NATURE STRUCTURAL & MOLECULAR BIOLOGY Gu, S., Jin, L., Zhang, F., Sarnow, P., Kay, M. A. 2009; 16 (2): 144-150


    MicroRNAs (miRNAs) interact with target sites located in the 3' untranslated regions (3' UTRs) of mRNAs to downregulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target-site localization in the 3' UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as small interfering RNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

    View details for DOI 10.1038/nsmb.1552

    View details for Web of Science ID 000263286600010

    View details for PubMedID 19182800

    View details for PubMedCentralID PMC2713750

  • MicroRNA-mediated gene silencing. Progress in molecular biology and translational science Pager, C. T., Wehner, K. A., Fuchs, G., Sarnow, P. 2009; 90: 187-210


    MicroRNAs are 20-21 nucleotides-long noncoding RNAs that function as posttranscriptional regulators of gene expression in a variety of organisms ranging from plants to mammalian cells. These regulators are encoded by approximately 800 genes in the mammalian genome and target half of the mRNAs in mammalian cells. While the biogenesis of microRNAs is fairly well understood, the mechanism by which target genes are regulated remains controversial. The recent discoveries that viruses encode microRNAs or subvert host cell microRNAs has enhanced our knowledge about biological functions of microRNAs during disease and has suggested that microRNAs could be used as targets in antiviral gene therapy. This review will provide a brief history of microRNA research, discuss the biogenesis and mechanisms of microRNAs, and summarize findings that have employed inhibitors of microRNA miR-122 to treat hepatitis C virus-induced liver disease.

    View details for DOI 10.1016/S1877-1173(09)90005-9

    View details for PubMedID 20374742

  • MicroRNA-Mediated Gene Silencing TRANSLATIONAL CONTROL IN HEALTH AND DISEASE Pager, C. T., Wehner, K. A., Fuchs, G., Sarnow, P. 2009; 90: 187-210
  • Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome CELL HOST & MICROBE Jopling, C. L., Schuetz, S., Sarnow, P. 2008; 4 (1): 77-85


    MicroRNAs usually interact with 3' noncoding regions (3'NCRs) of target mRNAs leading to downregulation of mRNA expression. In contrast, liver-specific microRNA miR-122 interacts with the 5' end of the hepatitis C virus RNA genome, resulting in increased viral RNA abundance. We find that inserting the viral miR-122 binding site into the 3' noncoding region of a reporter mRNA leads to downregulation of mRNA expression, indicating that the location of the miR-122 binding site dictates its effect on gene regulation. Furthermore, we discovered an adjacent, second miR-122 binding site, separated from the first by a highly conserved 14-nucleotide sequence. Mutational analysis demonstrates that both miR-122 binding sites in a single viral genome are occupied by the microRNA and function cooperatively to regulate target gene expression. These findings set a paradigm for dual, position-dependent functions of tandem microRNA-binding sites. Targeting an oligomeric microRNA complex offers potential as an antiviral-intervention strategy.

    View details for DOI 10.1016/j.chom.2008.05.013

    View details for Web of Science ID 000257908400010

    View details for PubMedID 18621012

    View details for PubMedCentralID PMC3519368

  • LNA-mediated microRNA silencing in non-human primates NATURE Elmen, J., Lindow, M., Schutz, S., Lawrence, M., Petri, A., Obad, S., Lindholm, M., Hedtjarn, M., Hansen, H. F., Berger, U., Gullans, S., Kearney, P., Sarnow, P., Straarup, E. M., Kauppinen, S. 2008; 452 (7189): 896-U10


    microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease and therefore represent a potential new class of targets for therapeutic intervention. Despite recent progress in silencing of miRNAs in rodents, the development of effective and safe approaches for sequence-specific antagonism of miRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that the simple systemic delivery of a unconjugated, PBS-formulated locked-nucleic-acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed miR-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg kg(-1) LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg kg(-1) LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring miRNA function in rodents and primates, and support the potential of these compounds as a new class of therapeutics for disease-associated miRNAs.

    View details for DOI 10.1038/nature06783

    View details for Web of Science ID 000255026000053

    View details for PubMedID 18368051

  • How viruses avoid stress CELL HOST & MICROBE Schutz, S., Sarnowl, P. 2007; 2 (5): 284-285


    Cellular responses to counter virus infection lead to the induction of cytoplasmic stress granules, which are composed of translationally stalled mRNAs. In this issue of Cell Host & Microbe, White and colleagues elucidate a mechanism where a poliovirus protease specifically cleaves a host cell factor involved in assembly of stress granules. This strategy ensures viral access to the limiting amounts of translation factors and interferes with host cell mRNA sorting.

    View details for DOI 10.1016/j.chom.2007.10.009

    View details for Web of Science ID 000251124300003

    View details for PubMedID 18005747

  • Inhibition of U snRNP assembly by a virus-encoded proteinase GENES & DEVELOPMENT Almstead, L. L., Sarnow, P. 2007; 21 (9): 1086-1097


    It has been proposed that defects in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes could account for the death of motor neurons in spinal muscular atrophy (SMA). We discovered that infection of cultured cells with poliovirus results in the specific cleavage of the host factor Gemin3 by a virus-encoded proteinase, 2A(pro). Gemin3 is a component of the macromolecular SMN complex that mediates assembly of U snRNP complexes by aiding the heptameric oligomerization of Sm proteins onto U snRNAs. Using in vitro Sm core assembly assays, we found that lowering the intracellular amounts of Gemin3 by either poliovirus infection or small interfering RNA (siRNA)-mediated knockdown of Gemin3 resulted in reduced assembly of U snRNPs. Immunofluorescence analyses revealed a specific redistribution of Sm proteins from the nucleoplasm to the cytoplasmic periphery of the nucleus in poliovirus-infected cells. We propose that defects in U snRNP assembly may be shared features of SMA and poliomyelitis.

    View details for DOI 10.1101/gad.1535607

    View details for Web of Science ID 000246154100009

    View details for PubMedID 17473171

    View details for PubMedCentralID PMC1855234

  • MicroRNAs: expression, avoidance and subversion by vertebrate viruses NATURE REVIEWS MICROBIOLOGY Sarnow, P., Jopling, C. L., Norman, K. L., Schutz, S., Wehner, K. A. 2006; 4 (9): 651-659


    MicroRNAs (miRNAs), which can be expressed in a cell-type and tissue-specific manner, can influence the activities of genes that control cell growth and differentiation. Viruses often have clear tissue tropisms, raising the possibility that cellular miRNAs might modulate their pathogenesis. In this Review, we discuss recent findings that some vertebrate viruses either encode miRNAs or subvert cellular miRNAs, and that these miRNAs participate in both the infectious and the latent phase of the viral life cycle.

    View details for Web of Science ID 000239877900010

    View details for PubMedID 16912711

  • Polypyrimidine tract binding protein regulates IRES-mediated gene expression during apoptosis MOLECULAR CELL Bushell, M., Stoneley, M., Kong, Y. W., Hamilton, T. L., Spriggs, K. A., Dobbyn, H. C., Qin, X., Sarnow, P., Willis, A. E. 2006; 23 (3): 401-412


    During apoptosis there is a substantial reduction in the rate of protein synthesis, and yet some mRNAs avoid this translational inhibition. To determine the impact that receptor-mediated cell death has on the translational efficiency of a large number of mRNAs, translational profiling was performed on MCF7 cells treated with the apoptosis-inducing ligand TRAIL. Our data indicate that approximately 3% of mRNAs remain associated with the polysomes in apoptotic cells, and genes that are involved in transcription, chromatin modification/remodeling, and the Notch signaling pathway are particularly prevalent among the mRNAs that evade translational inhibition. Internal ribosome entry segments (IRESs) were identified in several of the mRNAs that remained associated with the polysomes during apoptosis, and, importantly, these IRESs functioned efficiently in apoptotic cells. Finally, the data showed that polypyrimidine tract binding protein (PTB, a known IRES trans-acting factor or ITAF) is pivotal in regulating the apoptotic process by controlling IRES function.

    View details for DOI 10.1016/j.molcel.2006.06.012

    View details for Web of Science ID 000239805700011

    View details for PubMedID 16885029

  • Initiation factor-independent translation mediated by the hepatitis C virus internal ribosome entry site RNA-A PUBLICATION OF THE RNA SOCIETY Lancaster, A. M., Jan, E., Sarnow, P. 2006; 12 (5): 894-902


    The hepatitis C viral mRNA initiates translation using an internal ribosome entry site (IRES) located in the 5' noncoding region of the viral genome. At physiological magnesium ion concentrations, the HCV IRES forms a binary complex with the 40S ribosomal subunit, recruits initiation factor eIF3 and the ternary eIF2/GTP/Met-tRNA(i)Met complex, and joins 60S subunits to assemble translation-competent 80S ribosomes. Here we show that in the presence of 5 mM MgCl2, the HCV IRES can initiate translation by an alternative mechanism that does not require known initiation factors. Specifically, the HCV IRES was shown to initiate translation in a reconstituted system consisting only of purified 40S and 60S subunits, elongation factors, and aminoacylated tRNAs at high magnesium concentration. Analyses of assembled complexes supported a mechanism by which preformed 80S ribosomes can assemble directly on the HCV IRES at high cation concentrations. This mechanism is reminiscent of that employed by the divergent IRES elements in the Dicistroviridae, exemplified by the cricket paralysis virus, which mediates initiation of protein synthesis without initiator tRNA.

    View details for DOI 10.1261/rna.2342306

    View details for Web of Science ID 000237237500020

    View details for PubMedID 16556939

    View details for PubMedCentralID PMC1440913

  • Modulation of hepatitis C virus RNA by a liver-specific microRNA Experimental Biology 2006 Annual Meeting Sarnow, P., Jopling, C., Norman, K., Yi, M. K., Lemon, S. FEDERATION AMER SOC EXP BIOL. 2006: A1336–A1336
  • Interaction of viruses with the mammalian RNA interference pathway VIROLOGY Schuetz, S., Sarnow, P. 2006; 344 (1): 151-157


    It has been known for some time that plants and insects use RNA interference (RNAi) as nucleic acid-based immunity against viral infections. However, it was unknown whether mammalian cells employ the RNA interference pathway as an antiviral mechanism as well. Over the past years, it has become clear that a variety of viruses, first in plants but recently in insect and mammalian viruses as well, encode suppressors of the RNAi pathway arguing for an antiviral role of this machinery. More recent findings have revealed that certain viruses encode their own microRNAs or microRNA-like RNA molecules, which are processed by the mammalian RNAi machinery. Furthermore, host-encoded microRNAs have been shown to both silence and enhance intracellular levels of viral RNAs. These findings argue that interactions between the RNAi pathway and viral genomes can profoundly affect the outcomes of the viral life cycles and contribute to the pathogenic signatures of the infectious agents.

    View details for DOI 10.1016/j.virol.2005.09.034

    View details for Web of Science ID 000234611000018

    View details for PubMedID 16364746

  • Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNA miR-122 71st Cold Spring Harbor Symposium on Quantitative Biology Jopling, C. L., Norman, K. L., Sarnow, P. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 2006: 369–376


    microRNAs (miRNAs) are small RNAs that in general down-regulate the intracellular abundance and translation of target mRNAs. We noted that sequestration of liver-specific miR-122 by modified antisense oligonucleotides resulted in a dramatic loss of hepatitis C virus (HCV) RNA in cultured human liver cells. A binding site for miR-122 was predicted to reside close to the 5' end of the viral genome, and its functionality was tested by mutational analyses of the miRNA-binding site in viral RNA, resulting in reduced intracellular viral RNA abundance. Importantly, ectopic expression of miR-122 molecules that contained compensatory mutations restored viral RNA abundance, revealing a genetic interaction between miR-122 and the viral RNA genome. Studies with replication-defective viral RNAs demonstrated that miR-122 affected mRNA abundance by positively modulating RNA replication. In contrast, interaction of miR-122 with the 3'-noncoding region (3'NCR) of the cellular mRNA encoding the cationic amino acid transporter CAT-1 resulted in the down-regulation of CAT-1 protein abundance. These findings provide evidence that a specific miRNA can regulate distinct target mRNAs in both a positive and negative fashion. The positive role of miR-122 in viral replication suggests that this miRNA could be targeted for antiviral therapy.

    View details for Web of Science ID 000245962800048

    View details for PubMedID 17381319

  • Takeover of host ribosomes by divergent IRES elements BioScience 2005 Conference Sarnow, P., Cevallos, R. C., Jan, E. PORTLAND PRESS LTD. 2005: 1479–1482


    The ribosome is the macromolecular machinery in the host cell for which all viruses have to compete. Early in infection, the viral mRNAs have to compete with the host for both the ribosomes and for the limited pool of eukaryotic initiation factors that are needed to facilitate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition, certain viruses have evolved to recruit ribosomes to IRESs (internal ribosome entry sites), highly specialized RNA elements that are located at the 5'-end of the viral genomes. Here, we discuss how divergent IRES elements can recruit ribosomes and start protein synthesis with only a minimal set of eukaryotic translation initiation factors, and how this mode of translation initiation aids viral gene amplification during early onset of innate immune responses.

    View details for Web of Science ID 000233930800065

    View details for PubMedID 16246150

  • Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA SCIENCE Jopling, C. L., Yi, M. K., Lancaster, A. M., Lemon, S. M., Sarnow, P. 2005; 309 (5740): 1577-1581


    MicroRNAs are small RNA molecules that regulate messenger RNA (mRNA) expression. MicroRNA 122 (miR-122) is specifically expressed and highly abundant in the human liver. We show that the sequestration of miR-122 in liver cells results in marked loss of autonomously replicating hepatitis C viral RNAs. A genetic interaction between miR-122 and the 5' noncoding region of the viral genome was revealed by mutational analyses of the predicted microRNA binding site and ectopic expression of miR-122 molecules containing compensatory mutations. Studies with replication-defective RNAs suggested that miR-122 did not detectably affect mRNA translation or RNA stability. Therefore, miR-122 is likely to facilitate replication of the viral RNA, suggesting that miR-122 may present a target for antiviral intervention.

    View details for DOI 10.1126/science.1113329

    View details for Web of Science ID 000231715000054

    View details for PubMedID 16141076

  • Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition GENES & DEVELOPMENT Cherry, S., Doukas, T., Armknecht, S., Whelan, S., Wang, H., Sarnow, P., Perrimon, N. 2005; 19 (4): 445-452


    The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.

    View details for DOI 10.1101/gad.1267905

    View details for Web of Science ID 000227124400007

    View details for PubMedID 15713840

    View details for PubMedCentralID PMC548945

  • Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp JOURNAL OF VIROLOGY Cevallos, R. C., Sarnow, P. 2005; 79 (2): 677-683


    The Taura syndrome virus (TSV), a member of the Dicistroviridae family of viruses, is a single-stranded positive-sense RNA virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroviruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in cricket paralysis virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.

    View details for DOI 10.1128/JVI.79.2.677-683.2005

    View details for Web of Science ID 000226149700002

    View details for PubMedID 15613295

    View details for PubMedCentralID PMC538571

  • The arginine rich motif (ARM) in light chain 3 (LC3) of microtubule associated proteins 1A and 1B is required for protein-RNA interaction and mRNA translation 44th Annual Meeting of the American-Society-for-Cell-Biology Ying, L., Lau, A., Sarnow, P., RABINOVITCH, M. AMER SOC CELL BIOLOGY. 2004: 216A–216A
  • Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: The IRES functions as an RNA-Based translation factor CELL Spahn, C. M., Jan, E., Mulder, A., Grassucci, R. A., Sarnow, P., Frank, J. 2004; 118 (4): 465-475


    Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.

    View details for Web of Science ID 000223650900009

    View details for PubMedID 15315759

  • Translation inhibition during the induction of apoptosis: RNA or protein degradation? Focused Meeting of the Biochemical-Society Bushell, M., Stoneley, M., Sarnow, P., Willis, A. E. PORTLAND PRESS LTD. 2004: 606–610


    The induction of apoptosis leads to a substantial inhibition of protein synthesis. During this process changes to the translation-initiation factors, the ribosome and the cellular level of mRNA have been documented. However, it is by no means clear which of these events are necessary to achieve translational shutdown. In this article, we discuss modifications to the translational apparatus that occur during apoptosis and examine the potential contributions that they make to the inhibition of protein synthesis. Moreover, we present evidence that suggests that a global increase in the rate of mRNA degradation occurs before the caspase-dependent cleavage of initiation factors. Increased mRNA decay is temporally correlated with the shutdown of translation and therefore plays a major role in the inhibition of protein synthesis in apoptotic cells.

    View details for Web of Science ID 000223459900014

    View details for PubMedID 15270687

  • Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection JOURNAL OF VIROLOGY Graham, K. L., Gustin, K. E., RIVERA, C., Kuyumcu-Martinez, N. M., Choe, S. S., Lloyd, R. E., Sarnow, P., Utz, P. J. 2004; 78 (12): 6313-6321


    DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.

    View details for DOI 10.1128/JVI.78.12.6313-6321.2004

    View details for Web of Science ID 000221772000025

    View details for PubMedID 15163725

    View details for PubMedCentralID PMC416498

  • Preferential translation of internal ribosome entry site-containing mRNAs during the mitotic cycle in mammalian cells JOURNAL OF BIOLOGICAL CHEMISTRY Qin, X. L., Sarnow, P. 2004; 279 (14): 13721-13728


    A cell synchronization protocol was established in which global and individual mRNA translational efficiencies could be examined. While global translational efficiency was reduced in mitotic cells, approximately 3% of mRNAs remained predominantly associated with large polysomes during mitosis, as determined by cDNA microarray analyses. The 5'-non-coding regions of six mRNAs were shown to contain internal ribosome entry sites (IRES). However, not all known mRNAs that contain IRES elements were actively translated during mitosis, arguing that specific IRES sequences are differentially regulated during mitosis.

    View details for DOI 10.1074/jbc.M312854200

    View details for Web of Science ID 000220478500055

    View details for PubMedID 14739278

  • Divergent tRNA-like element supports initiation, elongation, and termination of protein biosynthesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jan, E., Kinzy, T. G., Sarnow, P. 2003; 100 (26): 15410-15415


    The cricket paralysis virus internal ribosome entry site (IRES) can, in the absence of canonical initiation factors and initiator tRNA (Met-tRNAi), occupy the ribosomal P-site and assemble 80S ribosomes. Here we show that the IRES assembles mRNA-80S ribosome complexes by recruitment of 60S subunits to preformed IRES-40S complexes. Addition of eukaryotic elongation factors eEF1A and eEF2 and aminoacylated elongator tRNAs resulted in the synthesis of peptides, implying that the IRES RNA itself mimics the function of Met-tRNAi in the P-site to trigger the first translocation step without peptide bond formation. IRES-80S complexes that contained a stop codon in the A-site recruited eukaryotic release factor eRF1, resulting in ribosome rearrangements in a surprisingly eEF2-dependent manner. Thus, this P-site-occupying IRES directs the assembly of 80S ribosomes, sets the translational reading frame, and mimics the functions of both Met-tRNAi and peptidyl tRNA to support elongation and termination.

    View details for DOI 10.1073/pnas.2535183100

    View details for Web of Science ID 000187554600025

    View details for PubMedID 14673072

    View details for PubMedCentralID PMC307581

  • Enterovirus 71 contains a type IIRES element that functions when eukaryotic initiation factor eIF4G is cleaved VIROLOGY Thompson, S. R., Sarnow, P. 2003; 315 (1): 259-266


    Human enterovirus 71 (EV71) is a member of the Enterovirus genus of the Picornaviridae family. Other members of this family utilize an unusual mechanism of translation initiation whereby ribosomes are recruited internally to the viral RNA by an internal ribosome site (IRES) located in their 5' noncoding regions (5' NCR). Using dicistronic reporter constructs, we demonstrate that the 5' NCRs of the 7423/MS/87 and BrCr strains of EV71 function as an IRES both in extracts and in cultured cells. Preincubation of translation extracts with purified coxsackievirus 2A protease cleaved elF4G, a component of the cap binding complex, resulting in a significant decrease in translation of capped mRNAs. In contrast, the translational efficiency of the EV71 IRES was enhanced under this condition, demonstrating that the EV71 IRES functions similar to other enterovirus IRES elements when components of the cap binding protein complex are cleaved. Finally, insertion of an upstream, out-of-frame start codon in the 5' NCR of the EV71 genome inhibited IRES activity, suggesting that EV71 can be classified as a type I IRES, in which ribosomes first bind upstream of the initiation codon and then scan the mRNA until an appropriate downstream AUG start codon is encountered and protein synthesis commences.

    View details for DOI 10.1016/S0042-6822(03)00544-0

    View details for Web of Science ID 000186373700024

    View details for PubMedID 14592777

  • Viral internal ribosome entry site elements: Novel ribosome-RNA complexes and roles in viral pathogenesis JOURNAL OF VIROLOGY Sarnow, P. 2003; 77 (5): 2801-2806
  • Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus JOURNAL OF MOLECULAR BIOLOGY Jan, E., Sarnow, P. 2002; 324 (5): 889-902


    The cricket paralysis virus (CrPV), a member of the CrPV-like virus family, contains a single positive-stranded RNA genome that encodes two non-overlapping open reading frames separated by a short intergenic region (IGR). The CrPV IGR contains an internal ribosomal entry site (IRES) that directs the expression of structural proteins. Unlike previously described IRESs, the IGR IRES initiates translation by recruiting 80S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a GCU alanine codon located in the A-site of the ribosome. Here, we have shown that a variety of mutations, designed to disrupt individually three pseudoknot (PK) structures and alter highly conserved nucleotides among the CrPV-like viruses, inhibit IGR IRES-mediated translation. By separating the steps of translational initiation into ribosomal recruitment, ribosomal positioning and ribosomal translocation, we found that the mutated IRES elements could be grouped into two classes. One class, represented by mutations in PKII and PKIII, bound 40S subunits with significantly reduced affinity, suggesting that PKIII and PKII are involved in the initial recruitment of the ribosome. A second class of mutations, exemplified by alterations in PKI, did not affect 40S binding but altered the positioning of the ribosome on the IRES, indicating that PKI is involved in the correct positioning of IRES-associated ribosomes. These results suggest that the IGR IRES has distinct pseudoknot-like structures that make multiple contacts with the ribosome resulting in initiation factor-independent recruitment and correct positioning of the ribosome on the mRNA.

    View details for DOI 10.1016/S0022-2836(02)01099-9

    View details for Web of Science ID 000179960200001

    View details for PubMedID 12470947

  • Cytoplasmic expression of mRNAs containing the internal ribosome entry site and 3 ' noncoding region of hepatitis C virus: Effects of the 3 ' leader on mRNA translation and mRNA stability JOURNAL OF VIROLOGY Kong, L. K., Sarnow, P. 2002; 76 (24): 12457-12462


    Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5' end of the mRNA, and the polyadenosine binding protein, bound to the 3'-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3'-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3'-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5' ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3' end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3' noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3'-end sequences of these chimeric RNAs were examined. It was found that the HCV 3' noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.

    View details for DOI 10.1128/JVI.76.24.12457-12462.2002

    View details for Web of Science ID 000179344800006

    View details for PubMedID 12438571

    View details for PubMedCentralID PMC136727

  • Inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus JOURNAL OF VIROLOGY Gustin, K. E., Sarnow, P. 2002; 76 (17): 8787-8796


    Nucleocytoplasmic trafficking pathways and the status of nuclear pore complex (NPC) components were examined in cells infected with rhinovirus type 14. A variety of shuttling and nonshuttling nuclear proteins, using multiple nuclear import pathways, accumulated in the cytoplasm of cells infected with rhinovirus. An in vitro nuclear import assay with semipermeabilized infected cells confirmed that nuclear import was inhibited and that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the NPC was prevented in rhinovirus-infected cells. The relocation of cellular proteins and inhibition of nuclear import correlated with the degradation of two NPC components, Nup153 and p62. The degradation of Nup153 and p62 was not due to induction of apoptosis, because p62 was not proteolyzed in apoptotic HeLa cells, and Nup153 was cleaved to produce a 130-kDa cleavage product that was not observed in cells infected with poliovirus or rhinovirus. The finding that both poliovirus and rhinovirus cause inhibition of nuclear import and degradation of NPC components suggests that this may be a common feature of the replicative cycle of picornaviruses. Inhibition of nuclear import is predicted to result in the cytoplasmic accumulation of a large number of nuclear proteins that could have functions in viral translation, RNA synthesis, packaging, or assembly. Additionally, inhibition of nuclear import also presents a novel strategy whereby cytoplasmic RNA viruses can evade host immune defenses by preventing signal transduction into the nucleus.

    View details for DOI 10.1128/JVI.76.17.8787-8796.2002

    View details for Web of Science ID 000177334900030

    View details for PubMedID 12163599

    View details for PubMedCentralID PMC136411

  • Hijacking the translation apparatus by RNA viruses JOURNAL OF CELL BIOLOGY Bushell, M., Sarnow, P. 2002; 158 (3): 395-399


    As invading viruses do not harbor functional ribosomes in their virions, successful amplification of the viral genomes requires that viral mRNAs compete with cellular mRNAs for the host cell translation apparatus. Several RNA viruses have evolved remarkable strategies to recruit the host translation initiation factors required for the first steps in translation initiation by host cell mRNAs. This review describes the ways that three families of RNA viruses effectively usurp limiting translation initiation factors from the host.

    View details for DOI 10.1083/jcb.200205044

    View details for Web of Science ID 000177329900003

    View details for PubMedID 12163463

    View details for PubMedCentralID PMC2173839

  • Ribosomal proteins mediate the hepatitis C virus IRES-HeLa 40S interaction RNA-A PUBLICATION OF THE RNA SOCIETY Otto, G. A., Lukavsky, P. J., Lancaster, A. M., Sarnow, P., Puglisi, J. D. 2002; 8 (7): 913-923


    Translation of the hepatitis C virus genomic RNA is mediated by an internal ribosome entry site (IRES). The 330-nt IRES RNA forms a binary complex with the small 40S ribosomal subunit as a first step in translation initiation. Here chemical probing and 4-thiouridine-mediated crosslinking are used to characterize the interaction of the HCV IRES with the HeLa 40S subunit. No IRES-18S rRNA contacts were detected, but several specific crosslinks to 40S ribosomal proteins were observed. The identity of the crosslinked proteins agrees well with available structural information and provides new insights into HCV IRES function. The protein-rich surface of the 40S subunit thus mediates the IRES-ribosome interaction.

    View details for DOI 10.1017/S1355838202022057

    View details for PubMedID 12166646

  • Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice MOLECULAR THERAPY McCaffrey, A. P., Hashi, K., Meuse, L., Shen, S. L., Lancaster, A. M., Lukavsky, P. J., Sarnow, P., Kay, M. A. 2002; 5 (6): 676-684


    Hepatitis C virus (HCV) is an RNA virus infecting 1 in every 40 people worldwide. Development of new therapeutics for treating HCV has been hampered by the lack of small-animal models. We have adapted existing hydrodynamic transfection methods to optimize the delivery of RNAs to the cytoplasm of mouse liver cells in vivo. Transfected HCV genomic RNA failed to replicate in mouse liver, suggesting a post-entry block to viral replication. Real-time imaging of HCV internal ribosome entry site (IRES) firefly luciferase reporter mRNA translation in living mice demonstrated that the HCV IRES was functional in mouse liver. We then used this system as a model for studying HCV RNA translation in mice. We compared translation by several mutant HCV IRES variants in cell lysates, cultured cells, and mouse liver. We measured the contribution to translation of a cap, HCV 3'-untranslated region (UTR), poly(A) tail, domains II, IIIb, IIIabc, IIIabcd, IIId, and the initiator codon. Efficient translation required a 3'-UTR in mice and HeLa cells, but not in rabbit reticulocyte lysates. Translational regulation of transfected RNAs was stringent in mice. The method we describe could be useful for studies in mice of antisense or ribozyme inhibitors targeting the IRES as well as other RNA biochemical studies in vivo.

    View details for DOI 10.1006/mthe.2002.0600

    View details for Web of Science ID 000176082700007

    View details for PubMedID 12027551

  • Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor eIF2 alpha JOURNAL OF BIOLOGICAL CHEMISTRY Fernandez, J., Yaman, I., Sarnow, P., Snider, M. D., Hatzoglou, M. 2002; 277 (21): 19198-19205


    Initiation of translation from most cellular mRNAs occurs via scanning; the 40 S ribosomal subunit binds to the m(7)G-cap and then moves along the mRNA until an initiation codon is encountered. Some cellular mRNAs contain internal ribosome entry sequences (IRESs) within their 5'-untranslated regions, which allow initiation independently of the 5'-cap. This study investigated the ability of cellular stress to regulate the activity of IRESs in cellular mRNAs. Three stresses were studied that cause the phosphorylation of the translation initiation factor, eIF2alpha, by activating specific kinases: (i) amino acid starvation, which activates GCN2; (ii) endoplasmic reticulum (ER) stress, which activates PKR-like ER kinase, PERK kinase; and (iii) double-stranded RNA, which activates double-stranded RNA-dependent protein kinase (PKR) by mimicking viral infection. Amino acid starvation and ER stress caused transient phosphorylation of eIF2alpha during the first hour of treatment, whereas double-stranded RNA caused a sustained phosphorylation of eIF2alpha after 2 h. The effects of these treatments on IRES-mediated initiation were investigated using bicistronic mRNA expression vectors. No effect was seen for the IRESs from the mRNAs for the chaperone BiP and the protein kinase Pim-1. In contrast, translation mediated by the IRESs from the cationic amino acid transporter, cat-1, and of the cricket paralysis virus intergenic region, were stimulated 3- to 10-fold by all three treatments. eIF2alpha phosphorylation was required for the response because inactivation of phosphorylation prevented the stimulation. It is concluded that cellular stress can stimulate translation from some cellular IRESs via a mechanism that requires the phosphorylation of eIF2alpha. Moreover, there are distinct regulatory patterns for different cellular mRNAs that contain IRESs within their 5'-untranslated regions.

    View details for DOI 10.1074/jbc.M201052200

    View details for Web of Science ID 000175975800123

    View details for PubMedID 11877448

  • New ways of initiating translation in eukaryotes? MOLECULAR AND CELLULAR BIOLOGY Schneider, R., Agol, V. I., Andino, R., Bayard, F., Cavener, D. R., Chappell, S. A., Chen, J. J., Darlix, J. L., Dasgupta, A., Donze, O., DUNCAN, R., Elroy-Stein, O., Farabaugh, P. J., Filipowicz, W., Gale, M., Gehrke, L., Goldman, E., Groner, Y., Harford, J. B., Hatzoglou, M., He, B., Hellen, C. U., Hentze, M. W., Hershey, J., Hershey, P., Hohn, T., Holcik, M., Hunter, C. P., Igarashi, K., Jackson, R., Jagus, R., Jefferson, L. S., Joshi, B., Kaempfer, R., Katze, M. G., Kaufman, R. J., Kiledjian, M., Kimball, S. R., KIMCHI, A., Kirkegaard, K., Koromilas, A. E., KRUG, R. M., Kruys, V., Lamphear, B. J., Lemon, S., Lloyd, R. E., Maquat, L. E., Martinez-Salas, E., Mathews, M. B., Mauro, V. P., Miyamoto, S., Mohr, I., Morris, D. R., Moss, E. G., NAKASHIMA, N., Palmenberg, A., Parkin, N. T., Pe'ery, T., Pelletier, J., Peltz, S., Pestova, T. V., Pilipenko, E. V., Prats, A. C., Racaniello, V., Read, G. S., Rhoads, R. E., Richter, J. D., Rivera-Pomar, R., Rouault, T., Sachs, A., Sarnow, P., Scheper, G. C., SCHIFF, L., Schoenberg, D. R., Semler, B. L., Siddiqui, A., Skern, T., Sonenberg, N., Sossin, W., Standart, N., Tahara, S. M., Thomas, A. A., Toulme, J. J., Wilusz, J., Wimmer, E., Witherell, G., Wormington, M. 2001; 21 (23): 8238-8241

    View details for Web of Science ID 000172059100034

    View details for PubMedID 11710333

    View details for PubMedCentralID PMC99989

  • Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Thompson, S. R., Gulyas, K. D., Sarnow, P. 2001; 98 (23): 12972-12977


    Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes. Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.

    View details for Web of Science ID 000172076800028

    View details for PubMedID 11687653

  • Internal ribosome entry sites in eukaryotic mRNA molecules GENES & DEVELOPMENT Hellen, C. U., Sarnow, P. 2001; 15 (13): 1593-1612

    View details for Web of Science ID 000169824600001

    View details for PubMedID 11445534

  • Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source MOLECULAR AND CELLULAR BIOLOGY Kuhn, K. M., DeRisi, J. L., Brown, P. O., Sarnow, P. 2001; 21 (3): 916-927


    The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.

    View details for Web of Science ID 000166353700022

    View details for PubMedID 11154278

    View details for PubMedCentralID PMC86682

  • Effects of poliovirus infection on nucleo-cytoplasmic trafficking and nuclear pore complex composition EMBO JOURNAL Gustin, K. E., Sarnow, P. 2001; 20 (1-2): 240-249


    Infection of eukaryotic cells with lytic RNA viruses results in extensive interactions of viral gene products with macromolecular pathways of the host, ultimately leading to death of the infected cells. We show here that infection of cells with poliovirus results in the cytoplasmic accumulation of a variety of shuttling and non-shuttling nuclear proteins that use multiple nuclear import pathways. In vitro nuclear import assays using semi-permeabilized infected cells confirmed that nuclear import was blocked and demonstrated that docking of nuclear import receptor-cargo complexes at the cytoplasmic face of the nuclear pore complex (NPC) was prevented. Analysis of components of the NPC revealed that two proteins, Nup153 and p62, were proteolyzed during poliovirus infection. These results suggest that the cytoplasmic relocalization of numerous cellular proteins is caused by the inhibition of multiple nuclear import pathways via alterations in NPC composition in poliovirus-infected cells. Blocking of nuclear import points to a novel strategy by which cytoplasmic RNA viruses can evade host immune defenses, by preventing signal transduction to the nucleus.

    View details for Web of Science ID 000166555700026

    View details for PubMedID 11226174

  • Initiator Met-tRNA-independent translation mediated by an internal ribosome entry site element in cricket paralysis virus-like insect viruses COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Jan, E., Thompson, S. R., Wilson, J. E., Pestova, T. V., Hellen, C. U., Sarnow, P. 2001; 66: 285-292

    View details for Web of Science ID 000180643700028

    View details for PubMedID 12762030

  • Structural and functional investigation of the hepatitis C virus IRES. Nucleic acids research. Supplement (2001) Puglisi, J. D., Kim, I., Lukavsky, P., Otto, G., Lancaster, A., Sarnow, P. 2001: 263-?

    View details for PubMedID 12836365

  • Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function NATURE STRUCTURAL BIOLOGY Lukavsky, P. J., Otto, G. A., Lancaster, A. M., Sarnow, P., Puglisi, J. D. 2000; 7 (12): 1105-1110


    Translation of the hepatitis C virus (HCV) polyprotein is initiated at an internal ribosome entry site (IRES) element in the 5' untranslated region of HCV RNA. The HCV IRES element interacts directly with the 40S subunit, and biochemical experiments have implicated RNA elements near the AUG start codon as required for IRES-40S subunit complex formation. The data we present here show that two RNA stem loops, domains IIId and IIIe, are involved in IRES-40S subunit interaction. The structures of the two RNA domains were solved by NMR spectroscopy and reveal structural features that may explain their role in IRES function.

    View details for PubMedID 11101890

  • Distinct mRNAs that encode La autoantigen are differentially expressed and contain internal ribosome entry sites JOURNAL OF BIOLOGICAL CHEMISTRY Carter, M. S., Sarnow, P. 2000; 275 (36): 28301-28307


    Analysis by reverse transcription-polymerase chain reaction has suggested the existence of at least two La autoantigen-encoding mRNAs that contain different 5' noncoding regions (NCRs) linked to the same La coding region (Troster, H., Metzger, T. E., Semsei, I., Schwemmle, M., Winterpacht, A., Zabel, B., and Bachmann, M. (1994) J. Exp. Med. 180, 2059-2067). La-encoding transcripts La1 and La1' contain 115- and 483-nucleotide 5' NCRs, respectively. To determine whether the various La transcripts are functional mRNAs, the expression and polysomal association of natural La1 and La1' RNAs were examined. Although La1 transcripts were ubiquitously expressed in human tissues, La1' transcripts were predominantly expressed in peripheral blood leukocytes, especially in B, T, and natural killer cells. Both La1 and La1' transcripts associated with polysomes in natural killer cells, suggesting that these transcripts were functional mRNAs. Upon activation of B cells with the mitogens phorbol 12-myristate 13-acetate and ionomycin, the amount of La1' mRNA, but not La1, declined. In contrast, after chemical activation of T cells, the amount of La 1 mRNA, but not La1', declined. The mechanism by which the La1 and La1' 5' NCRs initiate translation initiation was tested in cultured human HeLa cells and in two different in vitro translation systems. It was found that both 5' NCRs can mediate translation initiation by internal initiation. These findings indicate that the constitutive expression of La1 mRNA and the tissue-specific expression of La1' mRNA can both allow La protein synthesis under conditions when cap-dependent translation is compromised, such as inflammation, apoptosis, or certain viral infections.

    View details for Web of Science ID 000089197100102

    View details for PubMedID 10871624

  • Initiation of protein synthesis from the A site of the ribosome CELL Wilson, J. E., Pestova, T. V., Hellen, C. U., Sarnow, P. 2000; 102 (4): 511-520


    Positioning of the translation initiation complex on mRNAs requires interaction between the anticodon of initiator Met-tRNA, associated with eIF2-GTP and 40S ribosomal subunit, and the cognate start codon of the mRNA. We show that an internal ribosome entry site located in the genome of cricket paralysis virus can form 80S ribosomes without initiator Met-tRNA, eIF2, or GTP hydrolysis, with a CCU triplet in the ribosomal P site and a GCU triplet in the A site. P-site mutagenesis revealed that the P site was not decoded, and protein sequence analysis showed that translation initiates at the triplet in the A site. Translational initiation from the A site of the ribosome suggests that the repertoire of translated open reading frames in eukaryotic mRNAs may be greater than anticipated.

    View details for Web of Science ID 000088908000012

    View details for PubMedID 10966112

  • Regulation of host cell translation by viruses and effects on cell function CURRENT OPINION IN MICROBIOLOGY Thompson, S. R., Sarnow, P. 2000; 3 (4): 366-370


    Viruses have evolved a remarkable variety of strategies to modulate the host cell translation apparatus with the aim of optimizing viral mRNA translation and replication. Recent studies have revealed that modulation of both host and viral mRNA translation can be accomplished by selective alteration of translation factors in virus-infected cells. These findings provide new insights into the functioning of the translational apparatus in both uninfected and infected cells.

    View details for Web of Science ID 000088817300008

    View details for PubMedID 10972496

  • Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites MOLECULAR AND CELLULAR BIOLOGY Wilson, J. E., Powell, M. J., Hoover, S. E., Sarnow, P. 2000; 20 (14): 4990-4999


    Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5' end of the viral RNA and is more active than the IRES located at the 5' end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5' end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

    View details for Web of Science ID 000087820000004

    View details for PubMedID 10866656

  • Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johannes, G., Carter, M. S., Eisen, M. B., Brown, P. O., Sarnow, P. 1999; 96 (23): 13118-13123


    Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5' end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5' noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

    View details for Web of Science ID 000083649400028

    View details for PubMedID 10557283

    View details for PubMedCentralID PMC23910

  • Functional coupling between replication and packaging of poliovirus replicon RNA JOURNAL OF VIROLOGY Nugent, C. I., Johnson, K. L., Sarnow, P., Kirkegaard, K. 1999; 73 (1): 427-435


    Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687-1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726-1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated.

    View details for Web of Science ID 000077461700049

    View details for PubMedID 9847348

    View details for PubMedCentralID PMC103849

  • Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites RNA-A PUBLICATION OF THE RNA SOCIETY Johannes, G., Sarnow, P. 1998; 4 (12): 1500-1513


    Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.

    View details for Web of Science ID 000077366700004

    View details for PubMedID 9848649

  • Viral ribonucleoprotein complex formation and nucleolar-cytoplasmic relocalization of nucleolin in poliovirus-infected cells JOURNAL OF VIROLOGY Waggoner, S., Sarnow, P. 1998; 72 (8): 6699-6709


    The poliovirus 3' noncoding region (3'NCR) is involved in the efficient synthesis of viral negative-stranded RNA molecules. A strong interaction between a 105-kDa host protein and the wild-type 3'NCR, but not with a replication-defective mutant 3'NCR, was detected. This 105-kDa protein was identified as nucleolin which predominantly resides in the nucleolus and has been proposed to function in the folding of rRNA precursor molecules. A functional role for nucleolin in viral genome amplification was examined in a cell-free extract which has been shown to support the assembly of infectious virus from virion RNA. At early times of viral gene expression, extracts depleted of nucleolin produced less infectious virus than extracts depleted of fibrillarin, another resident of the nucleolus, indicating a functional role of nucleolin in the early stages of the viral life cycle in this in vitro system. Immunofluorescence analysis of uninfected and infected cells showed a nucleocytoplasmic relocalization of nucleolin, but not of fibrillarin, in poliovirus-infected cells. Relocalization of nucleolin was not simply a consequence of virally induced inhibition of translation or transcription, because inhibitors of translation or transcription did not induce nucleolar-cytoplasmic relocalization of nucleolin. These findings suggest a novel virus-induced mechanism by which certain nucleolar proteins are selectively redistributed in infected cells.

    View details for Web of Science ID 000074730200053

    View details for PubMedID 9658117

  • Internal ribosome entry sites tests with circular mRNAs. Methods in molecular biology (Clifton, N.J.) Chen, C. Y., Sarnow, P. 1998; 77: 355-363

    View details for PubMedID 9770681

  • In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Haller, A. A., Sarnow, P. 1997; 94 (16): 8521-8526


    Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues. The methylated guanosine residue closely resembles the 5' terminal cap structure present on all eukaryotic mRNA molecules. The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from Saccharomyces cerevisiae. These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.

    View details for Web of Science ID A1997XQ12400042

    View details for PubMedID 9238009

  • Location of the internal ribosome entry site in the 5' non-coding region of the immunoglobulin heavy-chain binding protein (BiP) mRNA: Evidence for specific RNA-protein interactions NUCLEIC ACIDS RESEARCH Yang, Q., Sarnow, P. 1997; 25 (14): 2800-2807


    The 220 nucleotide 5'non-coding region (5'NCR) of the human immunoglobulin heavy chain binding protein (BiP) mRNA contains an internal ribosome entry site (IRES) that mediates the translation of the second cistron in a dicistronic mRNA in cultured mammalian cells. In this study, experiments are presented that locate the IRES immediately upstream of the start-site AUG codon in the BiP mRNA. Furthermore, crosslinking of thiouridine-labeled BiP IRES-containing RNA to cellular proteins identified the specific binding of two proteins, p60 and p95, to the 3'half of the BiP 5'NCR. Interestingly, both p60 and p95 bound also specifically to several viral IRES elements. This correlation suggests that p60 and p95 could have roles in internal initiation of cellular and viral IRES elements.

    View details for Web of Science ID A1997XL84100016

    View details for PubMedID 9207027

  • Starting at the beginning, middle, and end: Translation initiation in eukaryotes CELL Sachs, A. B., Sarnow, P., Hentze, M. W. 1997; 89 (6): 831-838

    View details for Web of Science ID A1997XE35700003

    View details for PubMedID 9200601

  • Translation-competent extracts from Saccharomyces cerevisiae: Effects of L-A RNA, 5' cap, and 3' poly(A) tail on translational efficiency of mRNAs METHODS-A COMPANION TO METHODS IN ENZYMOLOGY Iizuka, N., Sarnow, P. 1997; 11 (4): 353-360


    Yeast genetics has proven fruitful in the identification of key players that are involved in translational initiation. However, the exact roles of many translation initiation factors in translation initiation remain unknown. This has been due to lack of a suitable in vitro translation system in which the mode of action of certain translation factors can be studied. This report describes the preparation of cell-free Saccharomyces cerevisiae lysates that can mediate the translation of exogenously added mRNAs. Optimal translation required the absence of viral L-A RNA in the lysate and the presence of both a 5' cap and a 3' poly(A) tail on the mRNAs. A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well. In addition, the yeast lysates mediated translational initiation through several viral internal ribosome entry sites, demonstrating that the yeast translation apparatus can perform internal initiation. Thus, these lysates may be useful in the biochemical analysis of cap-dependent and cap-independent translation events.

    View details for Web of Science ID A1997XT37800004

    View details for PubMedID 9126550

  • Evidence for involvement of trans-acting factors in selection of the AUG start codon during eukaryotic translational initiation MOLECULAR AND CELLULAR BIOLOGY McBratney, S., Sarnow, P. 1996; 16 (7): 3523-3534


    The molecular mechanism with which an appropriate AUG codon is selected as the start site for translational initiation by eukaryotic ribosomes is not known. By using a cell-free translation system, small RNA molecules containing single AUG codons, surrounded by various nucleotide sequences, were tested for their abilities to interfere with the translation of a reporter mRNA. RNAs containing the AUG in an ACCAUGG context (Kozak consensus sequence) were able to inhibit translation of the reporter mRNA. In contrast, RNAs containing the AUG in a less favorable context for start site selection (for example, CAGAUGG) had no effect on the translation of the reporter mRNA. The effect mediated by the ACCAUGC-containing RNAs was not due to sequestration of ribosomal subunits or to particular structural features in these RNAs. To identify potential trans-acting factors that might be preferentially bound by ACCAUGG-containing RNAs, ACCAUGG- and CAGAUGC-containing RNAs with a single 4-thiouridine residue at the AUG were incubated with partially fractionated extracts, and AUG-binding proteins were identified after irradiation of the complexes with UV light and subsequent analysis by gel electrophoresis. The analysis (of such complexes in competition experiments revealed that proteins, approximately 50 and 100 kDa in size, were found to bind directly at the AUG codon embedded in the ACCAUGG motif. One of these proteins has been identified as the La autoantigen. These findings indicate that trans-acting factors may play a role in AUG start site selection during translational initiation.

    View details for Web of Science ID A1996UT08600031

    View details for PubMedID 8668168



    The ribosome scanning model predicts that eukaryotic ribosomal 40S subunits enter all messenger RNAs at their 5' ends. Here, it is reported that eukaryotic ribosomes can initiate translation on circular RNAs, but only if the RNAs contain internal ribosome entry site elements. Long-repeating polypeptide chains were synthesized from RNA circles with continuous open reading frames. These results indicate that ribosomes can translate such RNA circles for multiple consecutive rounds and that the free 5' end of a messenger RNA is not necessarily the entry point for 40S subunits.

    View details for Web of Science ID A1995QU57200039

    View details for PubMedID 7536344

  • Cap-independent translation and internal initiation of translation in eukaryotic cellular mRNA molecules CAP-INDEPENDENT TRANSLATION Iizuka, N., Chen, C., Yang, Q., Johannes, G., Sarnow, P. 1995; 203: 155-177

    View details for Web of Science ID A1995BE51H00008

    View details for PubMedID 7555089



    Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts were cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus B1 conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.

    View details for Web of Science ID A1994PM68400031

    View details for PubMedID 7935446



    Translation of hepatitis C virus (HCV) RNA is initiated by cap-independent internal ribosome binding to the 5' noncoding region (NCR). To identify the sequences and structural elements within the 5' NCR of HCV RNA that contribute to the initiation of translation, a series of point mutations was introduced within this sequence. Since the pyrimidine-rich tract is considered a characteristic feature of picornavirus internal ribosome entry site (IRES) elements, our mutational analysis focused on two putative pyrimidine tracts (Py-I and Py-II) within the HCV 5' NCR. Translational efficiency of these mutant RNAs was examined by in vitro translation and after RNA transfection into liver-derived cells. Mutational analysis of Py-I (nucleotides 120 to 130), supported by compensatory mutants, demonstrates that the primary sequence of this motif is not important but that a helical structural element associated with this region is critical for HCV IRES function. Mutations in Py-II (nucleotides 191 to 199) show that this motif is dispensable for IRES function as well. Thus, the pyrimidine-rich tract motif, which is considered as an essential element of the picornavirus IRES elements, does not appear to be a functional component of the HCV IRES. Further, the insertional mutagenesis study suggests a requirement for proper spacing between the initiator AUG and the upstream structures of the HCV IRES element for internal initiation of translation.

    View details for Web of Science ID A1994PL73600054

    View details for PubMedID 7933114

  • Internal initiation of translation. Current opinion in cell biology McBratney, S., Chen, C. Y., Sarnow, P. 1993; 5 (6): 961-965


    Although the 5' cap-dependent scanning mechanism can account for the translational initiation of most mRNAs in eukaryotic cells, several viral and cellular mRNAs contain nucleotide sequences in their 5' non-coding regions that can mediate binding of ribosomes to the mRNA, regardless of the modification state of the 5' ends. During the past year, some nuclear proteins normally involved in RNA processing have been shown also to facilitate 'internal' ribosome binding. Unexpected dual functions have, therefore, been suggested for these RNA-binding proteins, in both RNA biogenesis in the nucleus and RNA translation in the cytoplasm.

    View details for PubMedID 8129948

  • Internal initiation of translation CURRENT OPINION IN CELL BIOLOGY McBratney, S., Chen, C. Y., Sarnow, P. 1993; 5 (6): 961-965


    The sequences in the plus-stranded poliovirus RNA genome that dictate the specific amplification of viral RNA in infected cells remain unknown. We have analyzed the structure of the 3' noncoding region of the viral genome by thermodynamic-based structure calculation and by chemical and enzymatic probing of in vitro-synthesized RNAs and provide evidence for the existence of an RNA pseudoknot structure in this region. To explore the functional significance of this structure, revertants of a mutant bearing a lesion in the proposed pseudoknot and exhibiting a temperature-sensitive defect in viral RNA synthesis were isolated and mapped. The results of this genetic analysis established a correlation between the structure of the 3' terminus of the viral RNA and its function in vivo in RNA amplification. Furthermore, phylogenetic analysis indicated that a similar structure could be formed in coxsackievirus B1, a related enterovirus, which further supports a role for the pseudoknot structure in viral RNA amplification in infected cells.

    View details for Web of Science ID A1993LB79400001

    View details for PubMedID 8388482



    The human hepatitis C virus (HCV) contains a long 5' noncoding region (5' NCR). Computer-assisted and biochemical analyses suggest that there is a complex secondary structure in this region that is comparable to the secondary structures that are found in picornaviruses (E.A. Brown, H. Zhang, L.-H. Ping, and S.M. Lemon, Nucleic Acids Res. 20:5041-5045, 1992). Previous in vitro studies suggest that the HCV 5' NCR plays an important role during translation (K. Tsukiyama-Kohara, N. Iizuka, M. Kohara, and A. Nomoto, J. Virol. 66:1476-1483, 1992). Dicistronic and monocistronic expression vectors, in vitro translation, RNA transfections, and deletion mutagenesis studies were utilized to demonstrate unambiguously that the HCV 5' NCR is involved in translational control. Our data strongly support the conclusion that an internal ribosome entry site exists within the 5' noncoding sequences proximal to the initiator AUG. Furthermore, our results suggest that the HCV genome is translated in a cap-independent manner and that the sequences immediately upstream of the initiator AUG are essential for internal ribosome entry site function during translation.

    View details for Web of Science ID A1993LB79400046

    View details for PubMedID 8388503

  • Gene regulation: translational initiation by internal ribosome binding. Current opinion in genetics & development Oh, S. K., Sarnow, P. 1993; 3 (2): 295-300


    During the past year, several examples of cellular mRNAs have been described in which translational initiation occurs by internal ribosome binding, a mechanism hitherto thought to be restricted to picornaviral RNAs. New insights into the molecular mechanism of internal ribosome entry have been provided by the structural and functional analyses of both the internal ribosome entry sites and the protein factors that stimulate translation mediated by these elements.

    View details for PubMedID 8504255



    Genetic and biochemical studies have revealed that the 5' noncoding region of poliovirus mediates translation of the viral mRNA by an unusual mechanism involving entry of ribosomes in internal sequences of mRNA molecules. We have found that mRNAs bearing the 5' noncoding region of poliovirus were translated at an enhanced rate in poliovirus-infected mammalian cells at a time when translation of cellular mRNAs was not yet inhibited. This translational enhancement of the polioviral 5' noncoding region was mediated by the expression of virus-encoded polypeptide 2A. This indicates that 2A is a multifunctional protein involved directly or indirectly in the activation of viral mRNA translation, in addition to its known roles in viral polyprotein processing and in inhibition of cellular protein synthesis. Thus, 2A represents an activator of translation of a viral mRNA that is translated by an internal ribosome binding mechanism. A likely consequence of this role of 2A is the efficient translation of viral mRNAs early in the infectious cycle, when host cell mRNAs can still compete with viral mRNAs for the host cell translation apparatus.

    View details for Web of Science ID A1992JW79800061

    View details for PubMedID 1332040



    The Antennapedia (Antp) homeotic gene of Drosophila melanogaster has two promoters, P1 and P2. The resulting Antp mRNAs contain 1512-nucleotide (P1) and 1727-nucleotide (P2) 5'-noncoding regions, composed of exons A, B, D, and E (P1) or exons C, D, and E (P2), respectively. Multiple AUG codons are present in exons A, B, and C. We have found that 252-nucleotide exon D, common to mRNAs from both transcription units and devoid of AUG codons, can mediate initiation of translation by internal ribosome binding in cultured cells. Many mRNAs in Drosophila contain long 5'-noncoding regions with apparently unused AUG codons, suggesting that internal ribosome binding may be a common mechanism of translational initiation, and possibly its regulation, in Drosophila.

    View details for Web of Science ID A1992JM56900005

    View details for PubMedID 1355457



    Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.

    View details for Web of Science ID A1992HD26500029

    View details for PubMedID 1310763



    We have used an RNA transfection assay to study the translation of cellular and viral mRNAs with and without 5'-terminal m7GpppG cap structures in human tissue culture cells. HeLa cells were transfected with in vitro-transcribed hybrid RNA molecules containing the 5' noncoding regions of either luciferase or poliovirus linked to the coding region of the firefly luciferase gene. Transcripts containing a capped luciferase 5' noncoding region produced luciferase, while similar uncapped transcripts did not. In contrast, transcripts containing a capped 5' noncoding region of poliovirus accumulated 10-fold-lower levels of luciferase than similar transcripts without a terminal cap structure. Inhibition of poliovirus mRNA translation by a 5'-terminal cap structure was not observed in in vitro translation systems. This finding indicates that factors involved in cap-independent translation of poliovirus RNA are quantitatively or qualitatively different in human tissue culture cells and in in vitro translation systems. Furthermore, this study emphasizes the importance of studying translational control of mRNAs in intact cells.

    View details for Web of Science ID A1991GL29600079

    View details for PubMedID 1656097



    A Robosome-scanning model has been proposed to explain the initiation of eukaryotic messenger RNAs in which binding of the 43S ternary ribosomal subunit near or at the 5' end of the mRNA is facilitated by an interaction between the methylated cap-structure at the end of the mRNA and the cap-binding protein complex eIF-4F. But picornaviral mRNAs do not have a 5' terminal cap structure and are translated by internal ribosome binding. A cellular mRNA, encoding the immunoglobulin heavy-chain binding protein, can be translated in poliovirus-infected cells at a time when cap-dependent translation of host cell mRNAs is inhibited. We report here that the 5' leader of the binding protein mRNA can directly confer internal ribosome binding to an mRNA in mammalian cells, indicating that translation initiation by an internal ribosome-binding mechanism is used by eukaryotic mRNAs.

    View details for Web of Science ID A1991GD80500067

    View details for PubMedID 1652694



    Many functions of the poliovirus genome in virally infected cells have been elucidated. However, the role of 2B (and of its precursor polypeptide, 2BC), encoded by the P2 region in the poliovirus genome, remains unknown. We have employed a genetic approach to examine the role of 2B in poliovirus-infected cells. We report here the phenotype of one previously isolated mutant in the 2B coding region, 2B201. In addition, we have constructed one additional mutation in the 2B coding region of an infectious poliovirus cDNA clone. Upon transfection into monkey Vero cells we could recover two 2B mutant polioviruses, 2B204 and 2B205. All three mutants exhibited small-plaque phenotypes on monkey Vero and human HeLa cells and displayed primary defects in viral RNA synthesis. None of the 2B mutants could be complemented by wild-type virus. Instead, the mutants exhibited a dosage-dependent dominance over wild-type poliovirus. Thus, the phenotypes of these 2B mutants implicate 2B and possibly its precursor, 2BC, in viral RNA amplification in poliovirus-infected cells, and the dominance of the 2B mutants suggests a structural role for 2B in viral replication complexes.

    View details for Web of Science ID A1991FY29700047

    View details for PubMedID 1649334



    Several mutations were introduced into an infectious poliovirus cDNA clone by inserting different oligodeoxynucleotide linkers into preexisting DNA restriction endonuclease sites in the viral cDNA. Ten mutated DNAs were constructed whose lesions mapped in the 5' noncoding region or in the capsid coding region of the viral genome. Eight of these mutated cDNAs did not give rise to infectious virus upon transfection into human cells, one yielded virus with a wild-type phenotype, and one gave rise to a viral mutant with a small-plaque phenotype. This last mutant, designated 1-5NC-S21, bears a 6-nucleotide insertion in the loop of a stable RNA hairpin at the very 5' end of the viral genome. Detailed analysis of the biological properties of 1-5NC-S21 showed that the primary defect in mutant-infected cells is a fivefold decrease in translation relative to wild-type-infected cells. Transfection into HeLa cells of in vitro-synthesized RNA molecules bearing either the 5' noncoding region of 1-5NC-S21 or wild-type poliovirus upstream of a luciferase reporter gene showed that the mutated RNA hairpin was responsible for the observed decrease in viral translation in mutant-infected cells and conferred this defect to heterologous RNAs. These findings indicate that an RNA hairpin located at the extreme 5' end of the viral RNA and highly conserved among enteroviruses and rhinoviruses profoundly affects the translation efficiency of poliovirus RNA in infected cells.

    View details for Web of Science ID A1991ET44500041

    View details for PubMedID 1846205



    Genetic and biochemical analyses of the 5' noncoding region of poliovirus have indicated the importance of this region in both translation and amplification of the viral RNA. The role of the cellular machinery required for these events is just beginning to be revealed. Using an RNA gel retention assay, we have identified a cellular 50-kDa protein that forms a specific complex with a stable stem-loop structure present in the viral 5' noncoding region. The formation of the RNA-protein complex is dependent on the availability of free sulfhydryl groups in the protein. The possible involvement of this RNA-protein complex in the regulation of viral gene expression is discussed.

    View details for Web of Science ID A1990DR71400054

    View details for PubMedID 2165605



    Translation of the mRNA encoding the immunoglobulin heavy-chain binding protein (BiP) is enhanced in poliovirus-infected cells at a time when translation of host cell mRNAs is inhibited. To test whether the mRNA of BiP is translated by internal ribosome binding, like picornaviral RNAs, we constructed plasmids for the expression of dicistronic hybrid RNAs containing the 5' noncoding region (5'NCR) of BiP as an intercistronic spacer element between two cistrons. Expression of these dicistronic mRNAs in mammalian cells resulted in efficient translation of both cistrons, demonstrating that the 5'NCR of BiP can confer internal ribosome binding to a heterologous RNA. This result suggests that the mRNA encoding BiP is bifunctional and can be translated by an internal ribosome-binding mechanism, in addition to the conventional cap-dependent scanning mechanism. This is the first demonstration of a cellular mRNA that can be translated by internal ribosome binding, and implies that this may be a mechanism for cellular translational regulation.

    View details for Web of Science ID A1990GQ28100026

    View details for PubMedID 2133656


    View details for Web of Science ID A1990DZ93800006

    View details for PubMedID 2169383



    All cellular cytoplasmic mRNAs carry a 7-methylguanylate cap attached to their 5' ends. This cap structure is recognized by cap-binding proteins that then direct the binding of ribosomal subunits to this 5'-end complex. Poliovirus, a plus-stranded RNA virus, interferes with this cellular translation process by proteolytically inactivating the cap-binding protein complex. Subsequently the viral mRNA can be translated by an initiation process in which ribosomes bind internally to the mRNA [Pelletier, J. & Sonenberg, N. (1988) Nature (London) 334, 320-325], obviating cap-dependent translation. At least one cellular mRNA, encoding a heat shock-like protein, glucose-regulated protein 78/immunoglobulin heavy-chain binding protein, has been discovered to be translated at an increased rate in poliovirus-infected cells at a time when the translation of other cellular mRNAs is inhibited. The glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA thus exemplifies a cellular mRNA that is translated at a specifically enhanced rate by an as-yet-unresolved cap-independent initiation process in cells when the cap-binding protein complex is not functional.

    View details for Web of Science ID A1989AH94900028

    View details for PubMedID 2548189



    It has been shown by van der Werf et al. (S. van der Werf, J. Bradley, E. Wimmer, F. W. Studier, and J. Dunn, Proc. Natl. Acad. Sci. USA 83:2330-2334, 1986) that in vitro synthesis of poliovirus RNA by T7 RNA polymerase gives rise to infectious RNA molecules; however, these molecules are only 5% as infectious as RNA isolated from virions. A plasmid, T7D-polio, was constructed that allows the in vitro synthesis of full-length RNA molecules with two additional guanine residues at the 5' end. However, T7D-polio differed from the construct of van der Werf et al. in that RNA transcribed from T7D-polio has an authentic 3' end, ending with only a polyadenine nucleotide sequence. Transfection of these RNA molecules into mammalian cells produced wild-type poliovirus with an efficiency similar to that of virion RNA. The use of this vector in the characterization of viral mutants in vivo and in vitro is discussed.

    View details for Web of Science ID A1989R376600061

    View details for PubMedID 2535751



    We constructed several well-defined mutations in the nonstructural portion of the poliovirus type I (Mahoney strain) genome by making small insertions in an infectious cDNA clone. The derived viral strains carrying the mutations exhibited a variety of distinct plaque phenotypes. Thus, we were able to examine genetic complementation between different pairs of mutants by comparing the yields of progeny virus in mixed and single infections. Two mutants bearing lesions in the 2A and 3A regions of the genome, which are defective in the inhibition of host cell translation and the synthesis of viral RNA, respectively, could be rescued efficiently by genetic complementation; three replication-deficient mutants containing insertions in the 2B, 3D (replicase), and 3'-untranslated regions could not. Both the 2A and 3A mutants could be rescued by each other and by all of the other mutants tested. Because yield enhancement was apparent well before the completion of a single infectious cycle, it is likely that complementation of both mutants involved early diffusion of functional products. These data provide the first unambiguous evidence that the nonstructural portion of the poliovirus genome contains multiple complementation groups. The data also suggest that certain nonstructural functions act only in cis.

    View details for Web of Science ID A1986E804600027

    View details for PubMedID 3023656



    We have constructed an 8-base-pair insertion mutation in the 3' noncoding region of an infectious poliovirus cDNA clone that gives rise to a temperature-sensitive RNA synthesis mutant upon transfection into mammalian cells. The mutated cDNA was used to establish a cell line that releases the mutant poliovirus in a temperature-dependent fashion, representing a unique persistent viral infection. A poliovirus mutant mapping in the noncapsid region of the viral genome can be complemented in this cell line, implying that the cell line expresses viral proteins at the nonpermissive temperature.

    View details for Web of Science ID A1986AZB0800010

    View details for PubMedID 3003739



    Four classes of monoclonal antibody-producing cell lines have been obtained that detect human cytomegalovirus virion structural proteins. These antibodies react with (1) a major outer membrane virion glycoprotein(s) gp58-gp130, whose molecular weight varies between strains of cytomegalovirus, (2) a phosphoprotein, pp71, localized inside the virion membrane, (3) a phosphorylated nucleocapsid protein, pp155, and (4) a virion-associated phosphoprotein, pp29. Polyclonal immune human sera react with a large number of virion proteins including those detected by these monoclonal antibodies. These monoclonal antibodies were employed in a radioimmune assay to detect low levels (6 X 10(3) PFU/ml) of human cytomegalovirus in solution and human urine. These antibodies were also employed in a fluorescent antibody format to identify cytomegalovirus-infected cells obtained from human urine and nasopharyngeal aspirates. These reagents provide useful tools for studying the molecular biology of virus replication, for diagnosing cytomegalovirus infections, and for studying virus latency and activation.

    View details for Web of Science ID A1984SE39300010

    View details for PubMedID 6322416



    In soluble protein extracts obtained from adenovirus productively infected cells, monoclonal antibodies directed against the early region 1B 58,000-dalton (E1B-58K) protein immunoprecipitated, in addition to this protein, a polypeptide of 25,000 molecular weight. An analysis of tryptic peptides derived from this 25K protein demonstrated that it was unrelated to the E1B-58K protein. The tryptic peptide maps of the 25K protein produced in adenovirus 5 (Ad5)-infected HeLa cells and BHK cells were identical, whereas Ad3-infected HeLa cells produced a different 25K protein. The viral origin of this 25K protein was confirmed by an amino acid sequence determination of five methionine residues in two Ad2 tryptic peptides derived from the 25K protein. The positions of these methionine residues in the 25K protein were compared with the nucleotide sequence of Ad2 and uniquely mapped the gene for this protein to early region 4, subregion 6 of the viral genome. A mutant of Ad5 was obtained (Ad5 dl342) which failed to produce detectable levels of the E1B-58K protein. In HeLa cells infected with this mutant, monoclonal antibodies directed against the E1B-58K protein failed to detect the associated 25K protein. In 293 cells infected with Ad5 dl342, which contain an E1B-58K protein encoded by the integrated adenovirus genome, the mutant produced an E4-25K protein which associated with the E1B-58K protein derived from the integrated genome. Extracts of labeled Ad5 dl342-infected HeLa cells (E1B-58K-) were mixed in vitro with extracts of unlabeled Ad5 wild type-infected HeLa cells or 293 cells (E1B-58K+). When the mixed extracts were incubated with the E1B-58K monoclonal antibody, a labeled E4-25K protein was coimmunoprecipitated. When extracts of Ad5 dl342-infected HeLa cells and uninfected HeLa cells (both E1B-58K-) were mixed, the E1B-58K monoclonal antibody failed to immunoselect the E4-25K protein. These data provide evidence that the E1B-58K antigen is physically associated with an E4-25K protein in productively infected cells. This is the same E1B-58K protein that was previously shown to be associated with the cellular p53 antigen in adenovirus-transformed cells.

    View details for Web of Science ID A1984SD16700009

    View details for PubMedID 6699935



    Polyadenylated RNA was isolated from fibroblast cultures infected with human cytomegalovirus (HCMV) strain AD169 during the late phase of viral replication. The RNA was selected by hybridization to a series of cosmid clones containing the entire viral genome in partially overlapping segments. Translation of this RNA in a reticulocyte cell-free system allowed the mapping of virus specific polypeptides. Nine polypeptides synthesized in vitro comigrated with major virion structural proteins. An in vitro-translated protein of 71 kDa was precipitated by a monoclonal antibody directed against the phosphorylated internal envelope protein of 71 kDa. The map coordinates of viral DNA coding for this phosphoprotein were localized by hybrid selection with subcloned DNA fragments, and the direction of transcription was determined by hybrid selection with single-stranded DNA cloned in bacteriophage vector M13mp9. An in vitro translation with size-fractionated RNA, combined with immunoprecipitation and Northern blot analyses, indicated that an mRNA of 4 kb encodes the 71-kDa phosphoprotein. An mRNA of the same size, map coordinates, and orientation was translated into an abundant 65-kDa polypeptide which had the same size as the major structural phosphoprotein of HCMV.

    View details for Web of Science ID A1984SP48600009

    View details for PubMedID 6324477



    Two hybridoma cell lines were obtained, A1 and B6, which produced monoclonal antibodies reacting with the 44,000-MW C-terminal domain of the adenovirus type 5 DNA-binding protein (DBP). Clone A1 antibodies reacted with the native form of the DBP, but failed to recognize this protein after denaturation (by exposure to sodium dodecylsulfate, or production of the DBP at 39.5 degrees by H5ts107, a temperature-sensitive DBP mutant). Clone B6 antibodies bound to both the native and denatured forms of the DBP. Immunofluorescent staining of wild-type-virus infected cells revealed the DBP located in discrete nuclear patches. A1 and B6 antibodies detected this patched localization of the DBP in nuclei of H5ts107-infected cells grown at 32 degrees. However, at the nonpermissive temperature of 39.5 degrees, A1 antibodies failed to detect the DBP, and B6 antibodies gave a uniform nuclear fluorescent distribution of the DBP. Thus the nuclear pattern of localization for the DBP synthesized by H5ts107 was temperature dependent in this mutant.

    View details for Web of Science ID A1983RE06000021

    View details for PubMedID 6310869



    R(ts107)202 is a host range temperature-conditional mutant of adenovirus type 5. This mutant is temperature sensitive for replication and plaquing in 293 cells but is temperature independent for growth and plaquing in HeLa cells (J. C. Nicolas, F. Suarez, A. J. Levine, and M. Girard (1981) Virology 108, 521-524). The mutant was isolated in HeLa cells as a temperature-independent revertant of the H5ts107 temperature-sensitive mutant that maps in the adenovirus DNA binding protein (DBP). The reasons for the temperature conditional phenotype of this mutant in 293 cells were investigated. The mutant synthesized an unstable DBP in both HeLa and 293 cells at 39 degrees. In 293 cells at 39 degrees, about two- to threefold less viral DNA was synthesized by r(ts107)202 as compared to Ad5wt. R(ts107)202 infected cells at 39 degrees produced normal (wild-type) amounts of all detectable late viral structural proteins. The mutant failed, however, to produce infectious virus or assemble virus particles in 293 cells at 39 degrees. The altered DBP may therefore play a role in the assembly of virus particles, either directly or indirectly via an altered DNA structure. The failure of r(ts107)202 to assemble virion particles in 293 cells at 39 degrees furthermore suggests that virus assembly is dependent upon cellular factors that differ in HeLa and 293 cell.

    View details for Web of Science ID A1983QM68100017

    View details for PubMedID 6302984


    View details for Web of Science ID A1982PV78800004

    View details for PubMedID 7166756


    View details for Web of Science ID A1982PB36000025

    View details for PubMedID 7048730


    View details for Web of Science ID A1982PP12600027

    View details for PubMedID 6815890



    The adenovirus E1b-58kd tumor antigen has been detected in a physical association with a 54 kilodalton cellular protein in adenovirus-transformed mouse cells. Antibody specific for the E1b-58kd protein coimmunoprecipitates a 54 kd protein from transformed, but not from productively infected, cells. Monoclonal antibody specific for the cellular 54 kd protein coimmunoprecipitates the adenovirus E1b-58kd protein from transformed cell extracts. The same or closely related cellular 54 kd protein, associated with the adenovirus E1b-58kd protein, was present in the SV40 large T antigen-54 kd complex previously detected in SV40-transformed mouse cells. The identity of the 54 kd protein is based on the immunological specificities of the anti-54 kd monoclonal antibodies and partial peptide maps of the 54 kd protein associated with the adenovirus and SV40 tumor antigens. The adenovirus E1b-58kd-54 kd complex, like the SV40 large T antigen-54 kd complex, is heterogeneous in size or mass. While all of the cellular 54 kd protein in the adenovirus-transformed cell extract is found in a complex with the E1b-58kd protein, some of the viral 58 kd antigen is detected in a form not associated with the 54 kd protein. The fact that the adenovirus and Sv40 tumor antigens, both required for transformation, can be found in physical association with the same cellular protein in a transformed cell is a good indication that these two diverse viral proteins share some common mechanisms or functions.

    View details for Web of Science ID A1982ND14100023

    View details for PubMedID 6277513



    A histone H4-specific methyltransferase was purified 80-100-fold from nuclei of calf lymphocytes and from calf thymus. Some biochemical properties of the enzyme are described. The enzyme transfers in vitro methyl groups from S-adenosylmethionine preferentially to the lysine residue 20 of histone H4. This is the major in vivo methylation site of H4. DNA-bound or nucleosomal H4 is not methylated in vitro. We have used methylated and unmodified H4 (in the presence of sufficient quantities of the other core histones) for nucleosome reconstitution in vitro and have not found significant differences in the efficiencies of assembly.

    View details for Web of Science ID A1981MN14000009

    View details for PubMedID 7284392