Academic Appointments

Administrative Appointments

  • Chair, Stanford University School of Medicine - Microbiology & Immunology (2006 - 2010)

Current Research and Scholarly Interests

For many subcellular viruses and parasites, RNA, not DNA, is the carrier of genetic information. This has several interesting consequences for the genetics and biology of the virus. Poliovirus serves as a model to increase our understanding of positive-strand RNA viruses for which no vaccine is available and which remain a significant health hazard: examples include other picornaviruses, such as rhinoviruses, coxsackieviruses and the deadly enterovirus 71 as well as more distantly related positive-strand RNA viruses such as hepatitis C and Dengue fever.

Questions currently under scrutiny are posed below, and discussed in greater detail in our web site.

1. How does the biochemistry of RNA-dependent RNA polymerases affect the biology of RNA viruses?

2. How are the membranous structures on which viral RNA replication complexes assemble form, and
from what intracellular organelles do they derive?

3. Why are the genetic properties of many RNA genomes different from DNA genomes? How does the error-prone nature of RNA-dependent RNA replication and the membrane association of the RNA replication complexes affect these genetic properties?

4. How does the inhibition of the protein secretory apparatus by the 3A and 2B proteins of picornaviruses such as poliovirus affect their pathogenesis? What would happen to the secretion of interferons, and to the presentation of antigens in the context of MHC class I molecules, if the host secretory pathway were not inhibited during infection by polioviruses, rhinoviruses and coxsackieviruses?

2014-15 Courses

Graduate and Fellowship Programs

All Publications

  • Escape of non-enveloped virus from intact cells VIROLOGY Bird, S. W., Kirkegaard, K. 2015; 479: 444-449
  • Nonlytic spread of naked viruses. Autophagy Bird, S. W., Kirkegaard, K. 2015; 11 (2): 430-431


    How do viruses spread from cell to cell? Enveloped viruses acquire their surrounding membranes by budding: either through the plasma membrane or an internal membrane of infected cells. Thus, a newly budded enveloped virus finds itself either in the extracellular milieu or in a lumenal compartment from which it can exit the cell by conventional secretion. On the other hand, naked viruses such as poliovirus, nodavirus, adenovirus, and SV40 lack an external membrane. They are simply protein-nucleic acid complexes within the cytoplasm or nucleus of the infected cell, and thus would seem to have no other exit route than cell lysis. We have presented the first documentation of nonlytic spread of a naked virus, and showed the interconnections between this event and the process or components of the autophagy pathway.

    View details for DOI 10.4161/15548627.2014.994372

    View details for PubMedID 25680079

  • Dominant Drug Targets Suppress the Emergence of Antiviral Resistance ELIFE Tanner, E. J., Liu, H., Oberste, M. S., Pallansch, M., Collett, M. S., Kirkegaard, K. 2014; 3
  • Nonlytic viral spread enhanced by autophagy components PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bird, S. W., Maynard, N. D., Covert, M. W., Kirkegaard, K. 2014; 111 (36): 13081-13086
  • The Ins and Outs of Viral Infection: Keystone Meeting Review VIRUSES-BASEL Bird, S. W., Kirkegaard, K., Agbandje-McKenna, M., Freed, E. O. 2014; 6 (9): 3652-3662

    View details for DOI 10.3390/v6093652

    View details for Web of Science ID 000343107100019

  • Double-membraned Liposomes Sculpted by Poliovirus 3AB Protein JOURNAL OF BIOLOGICAL CHEMISTRY Wang, J., Ptacek, J. B., Kirkegaard, K., Bullitt, E. 2013; 288 (38): 27287-27298


    Infection with many positive-strand RNA viruses dramatically remodels cellular membranes, resulting in the accumulation of double-membraned vesicles that resemble cellular autophagosomes. In this study, a single protein encoded by poliovirus, 3AB, is shown to be sufficient to induce the formation of double-membraned liposomes via the invagination of single-membraned liposomes. Poliovirus 3AB is a 109-amino acid protein with a natively unstructured N-terminal domain. HeLa cells transduced with 3AB protein displayed intracellular membrane disruption; specifically, the formation of cytoplasmic invaginations. The ability of a single viral protein to produce structures of similar topology to cellular autophagosomes should facilitate the understanding of both cellular and viral mechanisms for membrane remodeling.

    View details for DOI 10.1074/jbc.M113.498899

    View details for Web of Science ID 000330597300025

    View details for PubMedID 23908350

  • The NeST Long ncRNA Controls Microbial Susceptibility and Epigenetic Activation of the Interferon-gamma Locus CELL Gomez, J. A., Wapinski, O. L., Yang, Y. W., Bureau, J., Gopinath, S., Monack, D. M., Chang, H. Y., Brahic, M., Kirkegaard, K. 2013; 152 (4): 743-754


    Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST (nettoie Salmonella pas Theiler's [cleanup Salmonella not Theiler's]) is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-γ (IFN-γ) abundance in activated CD8(+) T cells, increased Theiler's virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.

    View details for DOI 10.1016/j.cell.2013.01.015

    View details for Web of Science ID 000314945600010

    View details for PubMedID 23415224

  • Inhibition of Cellular Autophagy Deranges Dengue Virion Maturation JOURNAL OF VIROLOGY Mateo, R., Nagamine, C. M., Spagnolo, J., Mendez, E., Rahe, M., Gale, M., Yuan, J., Kirkegaard, K. 2013; 87 (3): 1312-1321


    Autophagy is an important component of the innate immune response, directly destroying many intracellular pathogens. However, some pathogens, including several RNA viruses, subvert the autophagy pathway, or components of the pathway, to facilitate their replication. In the present study, the effect of inhibiting autophagy on the growth of dengue virus was tested using a novel inhibitor, spautin-1 (specific and potent autophagy inhibitor 1). Inhibition of autophagy by spautin-1 generated heat-sensitive, noninfectious dengue virus particles, revealing a large effect of components of the autophagy pathway on viral maturation. A smaller effect on viral RNA accumulation was also observed. Conversely, stimulation of autophagy resulted in increased viral titers and pathogenicity in the mouse. We conclude that the presence of functional autophagy components facilitates viral RNA replication and, more importantly, is required for infectious dengue virus production. Pharmacological inhibition of host processes is an attractive antiviral strategy to avoid selection of treatment-resistant variants, and inhibitors of autophagy may prove to be valuable therapeutics against dengue virus infection and pathogenesis.

    View details for DOI 10.1128/JVI.02177-12

    View details for Web of Science ID 000313558100003

  • Neuron-to-neuron transmission of alpha-synuclein fibrils through axonal transport ANNALS OF NEUROLOGY Freundt, E. C., Maynard, N., Clancy, E. K., Roy, S., Bousset, L., Sourigues, Y., Covert, M., Melki, R., Kirkegaard, K., Brahic, M. 2012; 72 (4): 517-524


    The lesions of Parkinson disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded ?-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous ?-synuclein to misfold. Here, we characterized and quantified the axonal transport of ?-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport.We grew primary cortical mouse neurons in microfluidic devices to separate somata from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent ?-synuclein fibrils and their transfer to second-order neurons.Fibrillar ?-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar ?-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts.These results support the hypothesis that the progression of Parkinson disease can be caused by neuron-to-neuron spread of ?-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be trans-synaptic gives hope that ?-synuclein fibrils could be intercepted by drugs during the extracellular phase of their journey.

    View details for DOI 10.1002/ana.23747

    View details for Web of Science ID 000310544900009

    View details for PubMedID 23109146

  • Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting MOLECULAR SYSTEMS BIOLOGY Maynard, N. D., Macklin, D. N., Kirkegaard, K., Covert, M. W. 2012; 8


    Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron-sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA(Lys) uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron-sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans.

    View details for DOI 10.1038/msb.2011.101

    View details for Web of Science ID 000299892400001

    View details for PubMedID 22294093

  • Interstitial Contacts in an RNA-Dependent RNA Polymerase Lattice JOURNAL OF MOLECULAR BIOLOGY Tellez, A. B., Wang, J., Tanner, E. J., Spagnolo, J. F., Kirkegaard, K., Bullitt, E. 2011; 412 (4): 737-750


    Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the "thumb" domain of one polymerase and the back of the "palm" domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, we used a hybrid approach of electron microscopic and biochemical evaluation of both wild-type and mutant viral polymerases to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should facilitate antiviral drug design and provide a precedent for other positive-strand RNA viruses.

    View details for DOI 10.1016/j.jmb.2011.07.053

    View details for Web of Science ID 000295496500016

    View details for PubMedID 21839092

  • 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 Web of Science ID 000275295900016

    View details for PubMedID 20169186

  • Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays RNA-A PUBLICATION OF THE RNA SOCIETY Spagnolo, J. F., Rossignol, E., Bullitt, E., Kirkegaard, K. 2010; 16 (2): 382-393


    Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase-polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.

    View details for DOI 10.1261/rna.1955410

    View details for Web of Science ID 000273868900015

    View details for PubMedID 20051491

  • Bypass Suppression of Small-Plaque Phenotypes by a Mutation in Poliovirus 2A That Enhances Apoptosis JOURNAL OF VIROLOGY Burgon, T. B., Jenkins, J. A., Deitz, S. B., Spagnolo, J. F., Kirkegaard, K. 2009; 83 (19): 10129-10139


    The rate of protein secretion in host cells is inhibited during infection with several different picornaviruses, with consequences likely to have significant effects on viral growth, spread, and pathogenesis. This Sin(+) (secretion inhibition) phenotype has been documented for poliovirus, foot-and-mouth disease virus, and coxsackievirus B3 and can lead to reduced cell surface expression of major histocompatibility complex class I and tumor necrosis factor receptor as well as reduced extracellular secretion of induced cytokines such as interleukin-6 (IL-6), IL-8, and beta interferon. The inhibition of protein secretion is global, affecting the movement of all tested cargo proteins through the cellular secretion apparatus. To test the physiological significance of the Sin(-) and Sin(+) phenotypes in animal models, Sin(-) mutant viruses are needed that fail to inhibit host protein secretion and also exhibit robust growth properties. To identify such Sin(-) mutant polioviruses, we devised a fluorescence-activated cell sorter-based screen to select virus-infected cells that nevertheless expressed newly synthesized surface proteins. After multiple rounds of selection, candidate Sin(-) mutant viruses were screened for genetic stability, increased secretion of cargo molecules and wild-type translation and growth properties. A newly identified Sin(-) mutant poliovirus that contained coding changes in nonstructural proteins 2A (N32D) and 2C (E253G) was characterized. In this virus, the 2C mutation is responsible for the Sin(-) phenotype and the 2A mutation suppresses a resulting growth defect by increasing the rate of cell death and therefore the rate of viral spread. The 2A-N32D suppressor mutation was not allele specific and, by increasing the rate of cellular apoptosis, affected a completely different pathway than the 2C-E253G Sin(-) mutation. Therefore, the 2A mutation suppresses the 2C-E253G mutant phenotype by a bypass suppression mechanism.

    View details for DOI 10.1128/JVI.00642-09

    View details for Web of Science ID 000269614300045

    View details for PubMedID 19625405

  • Role of Microtubules in Extracellular Release of Poliovirus JOURNAL OF VIROLOGY Taylor, M. P., Burgon, T. B., Kirkegaard, K., Jackson, W. T. 2009; 83 (13): 6599-6609


    Cellular autophagy, a process that directs cytosolic contents to the endosomal and lysosomal pathways via the formation of double-membraned vesicles, is a crucial aspect of innate immunity to many intracellular pathogens. However, evidence is accumulating that certain RNA viruses, such as poliovirus, subvert this pathway to facilitate viral growth. The autophagosome-like membranes induced during infection with wild-type poliovirus were found to be, unlike cellular autophagosomes, relatively immobile. Their mobility increased upon nocodazole treatment, arguing that vesicular tethering is microtubule dependent. In cells infected with a mutant virus that is defective in its interaction with the host cytoskeleton and secretory pathway, vesicle movement increased, indicating reduced tethering. In all cases, the release of tethering correlated with increased amounts of extracellular virus, which is consistent with the hypothesis that small amounts of cytosol and virus entrapped by double-membraned structures could be released via fusion with the plasma membrane. We propose that this extracellular delivery of cytoplasmic contents be termed autophagosome-mediated exit without lysis (AWOL). This pathway could explain the observed exit, in the apparent absence of cellular lysis, of other cytoplasmic macromolecular complexes, including infectious agents and complexes of aggregated proteins.

    View details for DOI 10.1128/JVI.01819-08

    View details for Web of Science ID 000267354100027

    View details for PubMedID 19369338

  • Subversion of the Cellular Autophagy Pathway by Viruses AUTOPHAGY IN INFECTION AND IMMUNITY Kirkegaard, K. 2009; 335: 323-333


    Autophagy is a cellular process that creates double-membraned vesicles, engulfs and degrades cytoplasmic material, and generates and recycles nutrients. A recognized participant in the innate immune response to microbial infection, a functional autophagic response can help to control the replication of many viruses. However, for several viruses, there is functional and mechanistic evidence that components of the autophagy pathway act as host factors in viral replicative cycles, viral dissemination, or both. Investigating the mechanisms by which viruses subvert or imitate autophagy, as well as the mechanisms by which they inhibit autophagy, will reveal cell biological tools and processes that will be useful for understanding the many functional ramifications of the double-membraned vesicle formation and cytosolic entrapment unique to the autophagy pathway.

    View details for DOI 10.1007/978-3-642-00302-8_16

    View details for Web of Science ID 000273774900016

    View details for PubMedID 19802573

  • Potential subversion of autophagosomal pathway by picornaviruses AUTOPHAGY Taylor, M. P., Kirkegaard, K. 2008; 4 (3): 286-289


    The RNA replication complexes of small positive-strand RNA viruses such as poliovirus are known to form on the surfaces of membranous vesicles in the cytoplasm of infected mammalian cells. These membranes resemble cellular autophagosomes in their double-membraned morphology, cytoplasmic lumen, lipid-rich composition and the presence of cellular proteins LAMP 1 and LC3. Furthermore, LC3 protein is covalently modified during poliovirus infection in a manner indistinguishable from that observed during bona fide autophagy. This covalent modification can also be induced by the expression of viral protein 2BC in isolation. However, differences between poliovirus-induced vesicles and autophagosomes also exist: the viral-induced membranes are smaller, at 200-400 nm in diameter, and can be induced by the combination of two viral proteins, termed 2BC and 3A. Experimental suppression of expression of proteins in the autophagy pathway was found to reduce viral yield, arguing that this pathway facilitates viral infection, rather than clearing it. We have hypothesized that, in addition to providing membranous surfaces for assembly of viral RNA replication complexes, double-membraned vesicles provide a topological mechanism to deliver cytoplasmic contents, including mature virus, to the extracellular milieu without lysing the cell.

    View details for Web of Science ID 000254477400006

    View details for PubMedID 18094610

  • Modification of cellular autophagy protein LC3 by poliovirus JOURNAL OF VIROLOGY Taylor, M. P., Kirkegaard, K. 2007; 81 (22): 12543-12553


    Poliovirus infection remodels intracellular membranes, creating a large number of membranous vesicles on which viral RNA replication occurs. Poliovirus-induced vesicles display hallmarks of cellular autophagosomes, including delimiting double membranes surrounding the cytosolic lumen, acquisition of the endosomal marker LAMP-1, and recruitment of the 18-kDa host protein LC3. Autophagy results in the covalent lipidation of LC3, conferring the property of membrane association to this previously microtubule-associated protein and providing a biochemical marker for the induction of autophagy. Here, we report that a similar modification of LC3 occurs both during poliovirus infection and following expression of a single viral protein, a stable precursor termed 2BC. Therefore, one of the early steps in cellular autophagy, LC3 modification, can be genetically separated from the induction of double-membraned vesicles that contain the modified LC3, which requires both viral proteins 2BC and 3A. The existence of viral inducers that promote a distinct aspect of the formation of autophagosome-like membranes both facilitates the dissection of this cellular process and supports the hypothesis that this branch of the innate immune response is directly subverted by poliovirus.

    View details for DOI 10.1128/JVI.00755-07

    View details for Web of Science ID 000254065400045

    View details for PubMedID 17804493

  • Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex JOURNAL OF CELL SCIENCE Beske, O., Reichelt, M., Taylor, M. P., Kirkegaard, K., Andino, R. 2007; 120 (18): 3207-3218


    Cells infected with poliovirus exhibit a rapid inhibition of protein secretion and disruption of the Golgi complex. Neither the precise step at which the virus inhibits protein secretion nor the fate of the Golgi complex during infection has been determined. We find that transport-vesicle exit from the endoplasmic reticulum (ER) and trafficking to the ER-Golgi intermediate compartment (ERGIC) are unaffected in the poliovirus-infected cell. By contrast, poliovirus infection blocks transport from the ERGIC to the Golgi complex. Poliovirus infection also induces fragmentation of the Golgi complex resulting in diffuse distribution of both large and small vesicles throughout the cell. Pre-treatment with nocodazole prevents complete fragmentation, indicating that microtubules are required for poliovirus-induced Golgi dispersion. However, virally induced inhibition of the secretory pathway is not affected by nocodazole, and Golgi dispersion was found to occur during infection with mutant viruses with reduce ability to inhibit protein secretion. We conclude that the dispersion of the Golgi complex is not in itself the cause of inhibition of traffic between the ERGIC and the Golgi. Instead, these phenomena are independent effects of poliovirus infection on the host secretory complex.

    View details for DOI 10.1242/jcs.03483

    View details for Web of Science ID 000249559400007

    View details for PubMedID 17711878

  • Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent RNA polymerase JOURNAL OF VIROLOGY Richards, O. C., Spagnolo, J. F., Lyle, J. M., Vleck, S. E., Kuchta, R. D., Kirkegaard, K. 2006; 80 (15): 7405-7415


    The 22-amino-acid protein VPg can be uridylylated in solution by purified poliovirus 3D polymerase in a template-dependent reaction thought to mimic primer formation during RNA amplification in infected cells. In the cell, the template used for the reaction is a hairpin RNA termed 2C-cre and, possibly, the poly(A) at the 3' end of the viral genome. Here, we identify several additional substrates for uridylylation by poliovirus 3D polymerase. In the presence of a 15-nucleotide (nt) RNA template, the poliovirus polymerase uridylylates other polymerase molecules in an intermolecular reaction that occurs in a single step, as judged by the chirality of the resulting phosphodiester linkage. Phosphate chirality experiments also showed that VPg uridylylation can occur by a single step; therefore, there is no obligatory uridylylated intermediate in the formation of uridylylated VPg. Other poliovirus proteins that could be uridylylated by 3D polymerase in solution were viral 3CD and 3AB proteins. Strong effects of both RNA and protein ligands on the efficiency and the specificity of the uridylylation reaction were observed: uridylylation of 3D polymerase and 3CD protein was stimulated by the addition of viral protein 3AB, and, when the template was poly(A) instead of the 15-nt RNA, the uridylylation of 3D polymerase itself became intramolecular instead of intermolecular. Finally, an antiuridine antibody identified uridylylated viral 3D polymerase and 3CD protein, as well as a 65- to 70-kDa host protein, in lysates of virus-infected human cells.

    View details for DOI 10.1128/JVI.02533-05

    View details for Web of Science ID 000239189100013

    View details for PubMedID 16840321

  • Bottleneck-mediated quasispecies restriction during spread of an RNA virus from inoculation site to brain PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pfeiffer, J. K., Kirkegaard, K. 2006; 103 (14): 5520-5525


    The amplification of RNA viruses such as poliovirus is associated with high error rates, and the resulting diversity likely facilitates viral survival within an infected host. However, within individual tissues of infected hosts, there may be barriers to viral spread that limit genome sampling. We tested whether poliovirus population diversity was maintained during viral spread to the brain of poliovirus receptor-expressing mice. Each of four restriction enzyme site-tagged viruses was shown to be able to replicate in the mouse brain. However, when infection was initiated by i.m., i.v., or i.p. routes, only a subset of the members of the injected pool was detectable in the brain. This jackpot effect was the result of a bottleneck in viral transit from the inoculation site to the brain. The bottleneck was difficult to overcome, requiring a 10(7) increase in viral inoculum to allow representation of all or most members of the infecting pool. Therefore, the bottleneck is not likely to be a physical barrier but an antiviral state induced by a founder virus. We suggest that the innate immune response can limit viral pathogenicity by limiting the number and therefore the diversity of viruses during spread to vulnerable tissues.

    View details for DOI 10.1073/pnas.0600834103

    View details for Web of Science ID 000236636400051

    View details for PubMedID 16567621

  • Nucleotide channel of RNA-dependent RNA polymerase used for intermolecular uridylylation of protein primer JOURNAL OF MOLECULAR BIOLOGY Tellez, A. B., Crowder, S., Spagnolo, J. F., Thompson, A. A., Peersen, O. B., Brutlag, D. L., Kirkegaard, K. 2006; 357 (2): 665-675


    Poliovirus VPg is a 22 amino acid residue peptide that serves as the protein primer for replication of the viral RNA genome. VPg is known to bind directly to the viral RNA-dependent RNA polymerase, 3D, for covalent uridylylation, yielding mono and di-uridylylated products, VPg-pU and VPg-pUpU, which are subsequently elongated. To model the docking of the VPg substrate to a putative VPg-binding site on the 3D polymerase molecule, we performed a variety of structure-based computations followed by experimental verification. First, potential VPg folded structures were identified, yielding a suite of predicted beta-hairpin structures. These putative VPg structures were then docked to the region of the polymerase implicated by genetic experiments to bind VPg, using grid-based and fragment-based methods. Residues in VPg predicted to affect binding were identified through molecular dynamics simulations, and their effects on the 3D-VPg interaction were tested computationally and biochemically. Experiments with mutant VPg and mutant polymerase molecules confirmed the predicted binding site for VPg on the back side of the polymerase molecule during the uridylylation reaction, opposite to that predicted to bind elongating RNA primers.

    View details for DOI 10.1016/j.jmb.2005.12.044

    View details for Web of Science ID 000236120200027

    View details for PubMedID 16427083

  • Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice PLOS PATHOGENS Pfeiffer, J. K., Kirkegaard, K. 2005; 1 (2): 102-110


    RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirus-receptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase.

    View details for DOI 10.1371/journal.ppat.0010011

    View details for Web of Science ID 000202893800002

    View details for PubMedID 16220146

  • Topology of double-membraned vesicles and the opportunity for non-lytic release of cytoplasm AUTOPHAGY Kirkegaard, K., Jackson, W. T. 2005; 1 (3): 182-184


    Infection of mammalian cells with several positive-strand RNA viruses induces double-membraned vesicles whose cytosolic surfaces serve as platforms for viral RNA replication. Our recent publication (Jackson et al. PLoS Biol 2005; 3:861-71) chronicled several similarities between poliovirus-induced membranes and autophagosomes, including induced co-localization of GFP-LC3 and LAMP1. Occasionally, the cytosolic lumen of these structures also contains viral particles; this likely results from wrapping of cytosol, which can contain high viral concentrations late in infection, by newly formed double membranes. Interestingly, RNAi treatment to reduce LC3 or Atg12p concentrations reduced yields of extracellular virus even more than intracellular virus. It is often assumed that exit of non-enveloped viruses such as poliovirus requires cell lysis. However, we hypothesize that autophagosome-like double-membranes, which can become single-membraned upon maturation, provide a long-sought mechanism for the observed non-lytic release of cytoplasmic viruses and possibly other cytoplasmic material resistant to the environment of maturing autophagosomes.

    View details for Web of Science ID 000238564100011

    View details for PubMedID 16874042

  • Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses NATURE GENETICS Crowder, S., Kirkegaard, K. 2005; 37 (7): 701-709


    The high error rates of viral RNA-dependent RNA polymerases create heterogeneous viral populations whose disparate RNA genomes affect each other's survival. We systematically screened the poliovirus genome and identified four sets of dominant mutations. Mutated alleles in capsid- and polymerase-coding regions resulted in dominant negative phenotypes, probably due to the proteins' oligomeric properties. We also identified dominant mutations in an RNA element required for priming RNA synthesis (CRE) and in the protein primer (VPg), suggesting that nonproductive priming intermediates are inhibitory. Mutations that inhibit the activity of viral proteinase 2A were dominant, arguing that inhibition of its known intramolecular activity creates a toxic product. Viral products that, when defective, dominantly interfere with growth of nondefective viruses will probably be excellent drug targets because drug-sensitive viruses should be dominant over drug-resistant variants. Accordingly, a virus sensitive to anticapsid compound WIN51711 dominantly inhibited the intracellular growth of a drug-resistant virus. Therefore, dominant inhibitor screening should validate or predict targets for antiviral therapy with reduced risk for drug resistance.

    View details for DOI 10.1038/ng1583

    View details for Web of Science ID 000230196400016

    View details for PubMedID 15965477

  • Inhibition of cellular protein secretion by picornaviral 3A proteins VIROLOGY Choe, S. S., Dodd, D. A., Kirkegaard, K. 2005; 337 (1): 18-29


    During poliovirus infection, anterograde traffic between the endoplasmic reticulum and the Golgi is inhibited due to the action of 3A, an 87 amino acid viral protein. The ability of poliovirus protein 3A to inhibit ER-to-Golgi traffic is not required for virus growth. Instead, we have suggested that the inhibition of host protein secretion, shown to reduce the secretion of interferon-beta, IL-6, and IL-8 and the expression of both newly synthesized MHC class I and TNF receptor in the plasma membrane of infected cells, affects growth in host organisms. To determine whether the ability of poliovirus 3A to inhibit ER-to-Golgi traffic is conserved, the ability of 3A proteins from several picornaviruses, including human rhinovirus 14, foot-and-mouth disease virus, enterovirus 71, hepatitis A, and Theiler's virus, was tested. Only the 3A proteins from another poliovirus, Sabin 3, and closely related coxsackievirus B3 inhibited ER-to-Golgi traffic as effectively as the 3A protein from poliovirus Mahoney type 1. Site-directed mutagenesis based on these findings and the three-dimensional structure of the amino-terminal domain of poliovirus 3A protein revealed that residues in the unstructured amino terminus of 3A are critical for the inhibition of host protein secretion.

    View details for DOI 10.1016/j.virol.2005.03.036

    View details for Web of Science ID 000229670700003

    View details for PubMedID 15914217

  • Allosteric effects of ligands and mutations on poliovirus RNA-dependent RNA polymerase JOURNAL OF VIROLOGY Boerner, J. E., Lyle, J. M., Daijogo, S., Semler, B. L., SCHULTZ, S. C., Kirkegaard, K., Richards, O. C. 2005; 79 (12): 7803-7811


    Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.

    View details for DOI 10.1128/JVI.79.12.7803-7811.2005

    View details for Web of Science ID 000229416100052

    View details for PubMedID 15919933

  • Subversion of cellular autophagosomal machinery by RNA viruses PLOS BIOLOGY Jackson, W. T., Giddings, T. H., Taylor, M. P., Mulinyawe, S., RABINOVITCH, M., Kopito, R. R., Kirkegaard, K. 2005; 3 (5): 861-871


    Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we show that several hallmarks of cellular autophagosomes can be identified in poliovirus-induced vesicles, including colocalization of LAMP1 and LC3, the human homolog of Saccharomyces cerevisiae Atg8p, and staining with the fluorophore monodansylcadaverine followed by fixation. Colocalization of LC3 and LAMP1 was observed early in the poliovirus replicative cycle, in cells infected with rhinoviruses 2 and 14, and in cells that express poliovirus proteins 2BC and 3A, known to be sufficient to induce double-membraned vesicles. Stimulation of autophagy increased poliovirus yield, and inhibition of the autophagosomal pathway by 3-methyladenine or by RNA interference against mRNAs that encode two different proteins known to be required for autophagy decreased poliovirus yield. We propose that, for poliovirus and rhinovirus, components of the cellular machinery of autophagosome formation are subverted to promote viral replication. Although autophagy can serve in the innate immune response to microorganisms, our findings are inconsistent with a role for the induced autophagosome-like structures in clearance of poliovirus. Instead, we argue that these double-membraned structures provide membranous supports for viral RNA replication complexes, possibly enabling the nonlytic release of cytoplasmic contents, including progeny virions, from infected cells.

    View details for DOI 10.1371/journal.pbio.0030156

    View details for Web of Science ID 000229125400014

    View details for PubMedID 15884975

  • Ribavirin resistance in hepatitis C virus replicon-containing cell lines conferred by changes in the cell line or mutations in the replicon RNA JOURNAL OF VIROLOGY Pfeiffer, J. K., Kirkegaard, K. 2005; 79 (4): 2346-2355


    Ribavirin (RBV), used in combination with alpha interferon to treat hepatitis C virus (HCV) infections, is a guanosine nucleotide analog that can increase the error rate of viral RNA-dependent RNA polymerases, imbalance intracellular nucleotide pools, and cause toxicity in many cell types. To determine potential mechanisms of RBV resistance during HCV RNA replication, we passaged HCV replicon-containing cell lines in the presence of increasing concentrations of RBV. RBV-resistant, HCV replicon-containing cell lines were generated, and the majority of RBV resistance was found to be conferred by changes in the cell lines. The resistant cell lines were defective in RBV import, as measured by [(3)H]RBV uptake experiments. These cell lines displayed reduced RBV toxicity and reduced error accumulation during infection with poliovirus, whose replication is known to be sensitive to RBV-induced error. For one RBV-resistant isolate, two mutations in the replicon RNA contributed to the observed phenotype. Two responsible mutations resided in the C-terminal region of NS5A, G404S, and E442G and were each sufficient for low-level RBV resistance. Therefore, RBV resistance in HCV replicon cell lines can be conferred by changes in the cell line or by mutations in the HCV replicon.

    View details for DOI 10.1128/JVI.79.4.2346-2355.2005

    View details for Web of Science ID 000226772100036

    View details for PubMedID 15681435

  • Intracellular topology and epitope shielding of poliovirus 3A protein JOURNAL OF VIROLOGY Choe, S. S., Kirkegaard, K. 2004; 78 (11): 5973-5982


    The poliovirus RNA replication complex comprises multiple viral and possibly cellular proteins assembled on the cytoplasmic surface of rearranged intracellular membranes. Viral proteins 3A and 3AB perform several functions during the poliovirus replicative cycle, including significant roles in rearranging membranes, anchoring the viral polymerase to these membranes, inhibiting host protein secretion, and possibly providing the 3B protein primer for RNA synthesis. During poliovirus infection, the immunofluorescence signal of an amino-terminal epitope of 3A-containing proteins is markedly shielded compared to 3A protein expressed in the absence of other poliovirus proteins. This is not due to luminal orientation of all or a subset of the 3A-containing polypeptides, as shown by immunofluorescence following differential permeabilization and proteolysis experiments. Shielding of the 3A epitope is more pronounced in cells infected with wild-type poliovirus than in cells with temperature-sensitive mutant virus that contains a mutation in the 3D polymerase coding region adjacent to the 3AB binding site. Therefore, it is likely that direct binding of the poliovirus RNA-dependent RNA polymerase occludes the amino terminus of 3A-containing polypeptides in the RNA replication complex.

    View details for DOI 10.1128/JVI.78.11.5973-5982.2004

    View details for Web of Science ID 000221513400046

    View details for PubMedID 15140995

  • Cellular autophagy: Surrender, avoidance and subversion by microorganisms NATURE REVIEWS MICROBIOLOGY Kirkegaard, K., Taylor, M. P., Jackson, W. T. 2004; 2 (4): 301-314

    View details for DOI 10.1038/nrmicro865

    View details for Web of Science ID 000220714800013

    View details for PubMedID 15031729

  • Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus JOURNAL OF VIROLOGY Konan, K. V., Giddings, T. H., Ikeda, M., Li, K., Lemon, S. M., Kirkegaard, K. 2003; 77 (14): 7843-7855


    The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.

    View details for DOI 10.1128/JVI.77.14.7843-7855.2003

    View details for Web of Science ID 000183899200016

    View details for PubMedID 12829824

  • A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pfeiffer, J. K., Kirkegaard, K. 2003; 100 (12): 7289-7294


    Ribavirin is a nucleotide analog that can be incorporated by viral polymerases, causing mutations by allowing base mismatches. It is currently used therapeutically as an antiviral drug during hepatitis C virus infections. During the amplification of poliovirus genomic RNA or hepatitis C replicons, error frequency is known to increase upon ribavirin treatment. This observation has led to the hypothesis that ribavirin's antiviral activity results from error catastrophe caused by increased mutagenesis of viral genomes. Here, we describe the generation of ribavirin-resistant poliovirus by serial viral passage in the presence of increasing concentrations of the drug. Ribavirin resistance can be caused by a single amino acid change, G64S, in the viral polymerase in an unresolved portion of the fingers domain. Compared with wild-type virus, ribavirin-resistant poliovirus displays increased fidelity of RNA synthesis in the absence of ribavirin and increased survival both in the presence of ribavirin and another mutagen, 5-azacytidine. Ribavirin-resistant poliovirus represents an unusual class of viral drug resistance: resistance to a mutagen through increased fidelity.

    View details for DOI 10.1073/pnas.1232294100

    View details for Web of Science ID 000183493500071

    View details for PubMedID 12754380

  • Visualization and functional analysis of RNA-dependent RNA polymerase lattices SCIENCE Lyle, J. M., Bullitt, E., Bienz, K., Kirkegaard, K. 2002; 296 (5576): 2218-2222


    Positive-strand RNA viruses such as poliovirus replicate their genomes on intracellular membranes of their eukaryotic hosts. Electron microscopy has revealed that purified poliovirus RNA-dependent RNA polymerase forms planar and tubular oligomeric arrays. The structural integrity of these arrays correlates with cooperative RNA binding and RNA elongation and is sensitive to mutations that disrupt intermolecular contacts predicted by the polymerase structure. Membranous vesicles isolated from poliovirus-infected cells contain structures consistent with the presence of two-dimensional polymerase arrays on their surfaces during infection. Therefore, host cytoplasmic membranes may function as physical foundations for two-dimensional polymerase arrays, conferring the advantages of surface catalysis to viral RNA replication.

    View details for Web of Science ID 000176379000058

    View details for PubMedID 12077417

  • Similar structural basis for membrane localization and protein priming by an RNA-dependent RNA polymerase JOURNAL OF BIOLOGICAL CHEMISTRY Lyle, J. M., Clewell, A., Richmond, K., Richards, O. C., Hope, D. A., SCHULTZ, S. C., Kirkegaard, K. 2002; 277 (18): 16324-16331


    Protein primers are used to initiate genomic synthesis of several RNA and DNA viruses, although the structural details of the primer-polymerase interactions are not yet known. Poliovirus polymerase binds with high affinity to the membrane-bound viral protein 3AB but uridylylates only the smaller peptide 3B in vitro. Mutational analysis of the polymerase identified four surface residues on the three-dimensional structure of poliovirus polymerase whose wild-type identity is required for 3AB binding. These mutants also decreased 3B uridylylation, arguing that the binding sites for the membrane tether and the protein primer overlap. Mutation of flanking residues between the 3AB binding site and the polymerase active site specifically decreased 3B uridylylation, likely affecting steps subsequent to binding. The physical overlap of sites for protein priming and membrane association should facilitate replication initiation in the membrane-associated complex.

    View details for DOI 10.1074/jbc.M112429200

    View details for Web of Science ID 000175510400143

    View details for PubMedID 11877407

  • Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase JOURNAL OF VIROLOGY Wang, Q. M., Hockman, M. A., Staschke, K., Johnson, R. B., Case, K. A., Lu, J. R., Parsons, S., Zhang, F. M., Rathnachalam, R., Kirkegaard, K., Colacino, J. M. 2002; 76 (8): 3865-3872


    The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, K(d), of approximately 22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 A. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.

    View details for DOI 10.1128/JVI.76.8.3865-3872.2002

    View details for Web of Science ID 000174520600029

    View details for PubMedID 11907226

  • Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection JOURNAL OF VIROLOGY Dodd, D. A., Giddings, T. H., Kirkegaard, K. 2001; 75 (17): 8158-8165


    During viral infections, the host secretory pathway is crucial for both innate and acquired immune responses. For example, the export of most proinflammatory and antiviral cytokines, which recruit lymphocytes and initiate antiviral defenses, requires traffic through the host secretory pathway. To investigate potential effects of the known inhibition of cellular protein secretion during poliovirus infection on pathogenesis, cytokine secretion from cells infected with wild-type virus and with 3A-2, a mutant virus carrying an insertion in viral protein 3A which renders the virus defective in the inhibition of protein secretion, was tested. We show here that cells infected with 3A-2 mutant virus secrete greater amounts of cytokines interleukin-6 (IL-6), IL-8, and beta interferon than cells infected with wild-type poliovirus. Increased cytokine secretion from the mutant-infected cells can be attributed to the reduced inhibition of host protein secretion, because no significant differences between 3A-2- and wild-type-infected cells were observed in the inhibition of viral growth, host cell translation, or the ability of wild-type- or 3A-2-infected cells to support the transcriptional induction of beta interferon mRNA. We surmise that the wild-type function of 3A in inhibiting ER-to-Golgi traffic is not required for viral replication in tissue culture but, by altering the amount of secreted cytokines, could have substantial effects on pathogenesis within an infected host. The global inhibition of protein secretion by poliovirus may reflect a general mechanism by which pathogens that do not require a functional protein secretory apparatus can reduce the native immune response and inflammation associated with infection.

    View details for Web of Science ID 000170343900040

    View details for PubMedID 11483761

  • Oligomeric structures of poliovirus polymerase are important for function EMBO JOURNAL Hobson, S. D., Rosenblum, E. S., Richards, O. C., Richmond, K., Kirkegaard, K., SCHULTZ, S. C. 2001; 20 (5): 1153-1163


    Central to the replication of poliovirus and other positive-strand RNA viruses is the virally encoded RNA-dependent RNA polymerase. Previous biochemical studies have suggested that direct polymerase- polymerase interactions might be important for polymerase function, and the structure of poliovirus polymerase has revealed two regions of extensive polymerase-polymerase interaction. To explore potential functional roles for the structurally observed polymerase-polymerase interactions, we have performed RNA binding and extension studies of mutant polymerase proteins in solution, disulfide cross-linking studies, mutational analyses in cells, in vitro activity analyses and RNA substrate modeling studies. The results of these studies indicate that both regions of polymerase-polymerase interaction observed in the crystals are indeed functionally important and, furthermore, reveal specific functional roles for each. One of the two regions of interaction provides for efficient substrate RNA binding and the second is crucial for forming catalytic sites. These studies strongly support the hypothesis that the polymerase- polymerase interactions discovered in the crystal structure provide an exquisitely detailed structural context for poliovirus polymerase function and for poliovirus RNA replication in cells.

    View details for Web of Science ID 000167393900022

    View details for PubMedID 11230138

  • MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Deitz, S. B., Dodd, D. A., Cooper, S., Parham, P., Kirkegaard, K. 2000; 97 (25): 13790-13795


    The effects of poliovirus 3A protein expression and poliovirus infection on the presentation of hepatitis C virus antigens in cultured chimpanzee cells were examined. Expression of poliovirus 3A protein inhibits protein secretion when expressed in isolation and was sufficient to protect chimpanzee cells from lysis by hepatitis C virus-specific cytotoxic T cells in standard (51)Cr-release assays. Poliovirus infection also inhibited antigen presentation, as determined by decreased cytotoxic T cell activation. A mutation in 3A that abrogates the inhibition of protein secretion also abolished the effects of poliovirus on antigen presentation. These results demonstrate that the inhibition of secretion observed in poliovirus-infected cells substantially reduces the presentation of new antigens on the cell surface. These observations may reflect a general mechanism by which nonenveloped viruses such as poliovirus and other viruses that do not require a functional protein secretory apparatus can evade detection by the cellular immune response.

    View details for Web of Science ID 000165728800061

    View details for PubMedID 11095746

  • Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles JOURNAL OF VIROLOGY Suhy, D. A., Giddings, T. H., Kirkegaard, K. 2000; 74 (19): 8953-8965


    All positive-strand RNA viruses of eukaryotes studied assemble RNA replication complexes on the surfaces of cytoplasmic membranes. Infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. Poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (ER), lysosomes, mitochondria, or the majority of Golgi-derived or endosomal membranes in buoyant density gradients, although changes in ionic strength affected ER and virus-induced vesicles, but not other cellular organelles, similarly. When expressed in isolation, two viral proteins of the poliovirus RNA replication complex, 3A and 2C, cofractionated with ER membranes. However, in cells that expressed 2BC, a proteolytic precursor of the 2B and 2C proteins, membranes identical in buoyant density to those observed during poliovirus infection were formed. When coexpressed with 2BC, viral protein 3A was quantitatively incorporated into these fractions, and the membranes formed were ultrastructurally similar to those in poliovirus-infected cells. These data argue that poliovirus-induced vesicles derive from the ER by the action of viral proteins 2BC and 3A by a mechanism that excludes resident host proteins. The double-membraned morphology, cytosolic content, and apparent ER origin of poliovirus-induced membranes are all consistent with an autophagic origin for these membranes.

    View details for Web of Science ID 000089244300020

    View details for PubMedID 10982339

  • 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

  • Complete protein linkage map of poliovirus P3 proteins: Interaction of polymerase 3D(pol) with VPg and with genetic variants of 3AB JOURNAL OF VIROLOGY Xiang, W. K., Cuconati, A., Hope, D., Kirkegaard, K., Wimmer, E. 1998; 72 (8): 6732-6741


    Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.

    View details for Web of Science ID 000074730200057

    View details for PubMedID 9658121

  • KH domain integrity is required for wild-type localization of Sam68 EXPERIMENTAL CELL RESEARCH McBride, A. E., Taylor, S. J., Shalloway, D., Kirkegaard, K. 1998; 241 (1): 84-95


    The protein Sam68 (Src-associated in mitosis, 68 kDa) has been found to bind to SH2 and to SH3 domain-containing proteins and to RNA. Although its protein-protein interactions implicate Sam68 in cell signaling, the significance of its RNA binding remains obscure. In most cells, Sam68 shows diffuse nucleoplasmic staining. Upon treatment with transcription inhibitors, however, Sam68 localize into punctate nuclear structures. Mutant forms of mouse Sam68 were overexpressed in human cells to test the importance of the KH domain, which is required for RNA binding, in the intracellular localization of Sam68. A small deletion within the KH domain (delta 206-218) or point mutation I184N had no effect upon the localization of overexpressed Sam68. Sam68 that contained a deletion of the entire KH domain (delta KH, delta 157-256) or point mutation G178E, however, localized to distinct nuclear spots. Furthermore, delta KH Sam68, unlike wild-type Sam68 and several other mutant Sam68 proteins, did not relocalize upon poliovirus infection and caused the normally cytoplasmic viral polymerase to localize to the nuclear spots. Thus both ongoing transcription and an intact KH domain are crucial determinants of the dynamic intracellular localization of Sam68.

    View details for Web of Science ID 000074191200009

    View details for PubMedID 9633516

  • Site size of cooperative single-stranded RNA binding by poliovirus RNA-dependent RNA polymerase JOURNAL OF BIOLOGICAL CHEMISTRY Beckman, M. T., Kirkegaard, K. 1998; 273 (12): 6724-6730


    The poliovirus RNA-dependent RNA polymerase binds cooperatively to single-stranded RNA. We have determined the minimal RNA-binding site size of the poliovirus polymerase using binding titration with oligonucleotides of increasing length. A dramatic increase in affinity was observed when the length of the oligo(U) increased from 8 to 10 nucleotides (nt), arguing that the minimal size of RNA for polymerase binding is 10 nt. Another increase in affinity seen as the oligo(U) reached 24 nt suggests that a 24-nucleotide RNA can be occupied by two polymerase molecules. Direct binding of wild-type polymerase to oligo(U)12 and oligo(U)24 RNAs showed differences in affinity and cooperativity consistent with this model. The increase in binding affinity seen for oligo(U)10 suggests either that the RNA-binding determinants are widely spaced on the polymerase structure or that a substantial conformational change in the polymerase occurs upon the filling of its RNA-binding site.

    View details for Web of Science ID 000072775900021

    View details for PubMedID 9506971

  • Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: Genetic and ultrastructural analysis JOURNAL OF VIROLOGY Doedens, J. R., Giddings, T. H., Kirkegaard, K. 1997; 71 (12): 9054-9064


    Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER.

    View details for Web of Science ID A1997YF89600012

    View details for PubMedID 9371562

  • Poliovirus RNA recombination in cell-free extracts RNA-A PUBLICATION OF THE RNA SOCIETY Tang, R. S., Barton, D. J., Flanegan, J. B., Kirkegaard, K. 1997; 3 (6): 624-633


    Poliovirus RNA has been shown to undergo homologous genetic recombination at a high frequency in infected human cells. Recently it has become possible to mimic the entire intracellular replicative cycle of poliovirus replication in cytoplasmic extracts prepared from HeLa cells, resulting in the generation of infectious poliovirions. The mechanism of poliovirus RNA recombination has been shown previously to be coupled to RNA replication, presumably by template switching during the replication of parental RNAs. Experiments were designed to test whether recombinant poliovirus RNA molecules are produced in a cell-free environment. Recombinant molecules generated bear marker sequences that can be detected physically by reverse transcription and PCR. We report here successful detection of poliovirus RNA recombination in a cell-free replication system. The frequency measured for cell-free RNA recombination between two polymorphic marker loci 656 nt apart was between 10(-2) and 10(-3) recombinants/genome, a frequency comparable to or slightly higher than that measured for RNA recombination in infected cells.

    View details for Web of Science ID A1997XB20800008

    View details for PubMedID 9174097

  • Cellular origin and ultrastructure of membranes induced during poliovirus infection JOURNAL OF VIROLOGY Schlegel, A., Giddings, T. H., Ladinsky, M. S., Kirkegaard, K. 1996; 70 (10): 6576-6588


    Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes.

    View details for Web of Science ID A1996VG12700006

    View details for PubMedID 8794292



    Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.

    View details for Web of Science ID A1995QM73300005

    View details for PubMedID 7889939



    We examined the importance of two interactions between poliovirus and its host cell: the putative association between viral proteins and a rearranged intermediate filament (IF) network and the apparent requirement for functional vesicle budding machinery within the host-cell secretory pathway. Poliovirus capsid proteins appeared to associate with reorganized IF proteins during infection. Treatment of cells with cytochalasin D and nocodazole in combination disrupted normal cytoskeletal organization and prevented the poliovirus-induced redistribution of IF proteins to a juxtanuclear location. However, this treatment had no effect on viral yields from single-cycle infections, indicating that neither cytoskeletal integrity nor a specific poliovirus-induced rearrangement of IF proteins is required in the poliovirus life cycle. In contrast, we report that the inhibition of poliovirus replication by brefeldin A (BFA), an inhibitor of secretory membrane traffic, is specific to the host cell. Polioviral yields were not affected by BFA in two BFA-resistant cell lines, demonstrating that BFA targets a host protein or process required by poliovirus. No BFA-resistant virus was detected in these experiments, further supporting the hypothesis that poliovirus replication requires secretory pathway function, perhaps for the generation of vesicles on which viral RNA replication complexes are assembled.

    View details for Web of Science ID A1994NQ96300017

    View details for PubMedID 8032247