Stanford Advisors


All Publications


  • The transcriptional landscape of Venezuelan equine encephalitis virus (TC-83) infection. PLoS neglected tropical diseases Yao, Z. n., Zanini, F. n., Kumar, S. n., Karim, M. n., Saul, S. n., Bhalla, N. n., Panpradist, N. n., Muniz, A. n., Narayanan, A. n., Quake, S. R., Einav, S. n. 2021; 15 (3): e0009306

    Abstract

    Venezuelan Equine Encephalitis Virus (VEEV) is a major biothreat agent that naturally causes outbreaks in humans and horses particularly in tropical areas of the western hemisphere, for which no antiviral therapy is currently available. The host response to VEEV and the cellular factors this alphavirus hijacks to support its effective replication or evade cellular immune responses are largely uncharacterized. We have previously demonstrated tremendous cell-to-cell heterogeneity in viral RNA (vRNA) and cellular transcript levels during flaviviral infection using a novel virus-inclusive single-cell RNA-Seq approach. Here, we used this unbiased, genome-wide approach to simultaneously profile the host transcriptome and vRNA in thousands of single cells during infection of human astrocytes with the live-attenuated vaccine strain of VEEV (TC-83). Host transcription was profoundly suppressed, yet "superproducer cells" with extremely high vRNA abundance emerged during the first viral life cycle and demonstrated an altered transcriptome relative to both uninfected cells and cells with high vRNA abundance harvested at later time points. Additionally, cells with increased structural-to-nonstructural transcript ratio exhibited upregulation of intracellular membrane trafficking genes at later time points. Loss- and gain-of-function experiments confirmed pro- and antiviral activities in both vaccine and virulent VEEV infections among the products of transcripts that positively or negatively correlated with vRNA abundance, respectively. Lastly, comparison with single cell transcriptomic data from other viruses highlighted common and unique pathways perturbed by infection across evolutionary scales. This study provides a high-resolution characterization of the VEEV (TC-83)-host interplay, identifies candidate targets for antivirals, and establishes a comparative single-cell approach to study the evolution of virus-host interactions.

    View details for DOI 10.1371/journal.pntd.0009306

    View details for PubMedID 33788849

  • Nonproteolytic K29-Linked Ubiquitination of the PB2 Replication Protein of Influenza A Viruses by Proviral Cullin 4-Based E3 Ligases MBIO Karim, M., Biquand, E., Declercq, M., Jacob, Y., van der Werf, S., Demeret, C. 2020; 11 (2)

    Abstract

    The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.

    View details for DOI 10.1128/mBio.00305-20

    View details for Web of Science ID 000531071300053

    View details for PubMedID 32265326

    View details for PubMedCentralID PMC7157767

  • Influenza A virus co-opts ERI1 exonuclease bound to histone mRNA to promote viral transcription Nucleic Acids Research Declercq, M., Biquand, E., Karim, M., Pietrosemoli, N., Jacob, Y., Demeret, C., Barbezange, C., Werf, S. v. 2020

    View details for DOI 10.1093/nar/gkaa771

  • BIKE regulates dengue virus infection and is a cellular target for broad-spectrum antivirals. Antiviral research Pu, S., Schor, S., Karim, M., Saul, S., Robinson, M., Kumar, S., Prugar, L. I., Dorosky, D. E., Brannan, J., Dye, J. M., Einav, S. 2020: 104966

    Abstract

    Global health is threatened by emerging viruses, many of which lack approved therapies and effective vaccines, including dengue, Ebola, and Venezuelan equine encephalitis. We previously reported that AAK1 and GAK, two of the four members of the understudied Numb-associated kinases (NAK) family, control intracellular trafficking of RNA viruses. Nevertheless, the role of BIKE and STK16 in viral infection remained unknown. Here, we reveal a requirement for BIKE, but not STK-16, in dengue virus (DENV) infection. BIKE mediates both early (postinternalization) and late (assembly/egress) stages in the DENV life cycle, and this effect is mediated in part by phosphorylation of a threonine 156 (T156) residue in the mu subunit of the adaptor protein (AP) 2 complex. Pharmacological compounds with potent anti-BIKE activity, including the investigational anticancer drug 5Z-7-oxozeaenol and more selective inhibitors, suppress DENV infection both in vitro and ex vivo. BIKE overexpression reverses the antiviral activity, validating that the mechanism of antiviral action is, at least in part, mediated by BIKE. Lastly, 5Z-7-oxozeaenol exhibits antiviral activity against viruses from three unrelated RNA viral families with a high genetic barrier to resistance. These findings reveal regulation of poorly understood stages of the DENV life cycle via BIKE signaling and establish a proof-of-principle that pharmacological inhibition of BIKE can be potentially used as a broad-spectrum strategy against acute emerging viral infections.

    View details for DOI 10.1016/j.antiviral.2020.104966

    View details for PubMedID 33137362

  • Influenza A virus co-opts ERI1 exonuclease bound to histone mRNA to promote viral transcription NUCLEIC ACIDS RESEARCH Declercq, M., Biquand, E., Karim, M., Pietrosemoli, N., Jacob, Y., Demeret, C., Barbezange, C., van der Werf, S. 2020; 48 (18): 10428–40

    Abstract

    Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus.

    View details for DOI 10.1093/nar/gkaa771

    View details for Web of Science ID 000593122700033

    View details for PubMedID 32960265

    View details for PubMedCentralID PMC7544206

  • In silico analysis revealed Zika virus miRNAs associated with viral pathogenesis through alteration of host genes involved in immune response and neurological functions JOURNAL OF MEDICAL VIROLOGY Islam, M., Khan, M., Murad, M., Karim, M., Islam, A. 2019; 91 (9): 1584–94

    Abstract

    The concurrent Zika Virus (ZIKV) outbreaks in the United States and Northeast Brazil have evoked global surveillance. Zika infection has been correlated with severe clinical symptoms, such as microcephaly, Guillain-Barré syndrome, and other congenital brain abnormalities. Recent data suggest that ZIKV predominantly targets neural progenitor cells leading to neurological impairment. Despite the clinical evidence, detailed experimental mechanism of ZIKV neurotropic pathogenesis has not been fully understood yet. Here we hypothesized that ZIKV produces miRNAs, which target essential host genes involved in various cellular pathways facilitating their survival through immune evasion and progression of disease during brain development.From genome sequence information using several bioinformatic tools, we predicted pri-miRNAs, pre-miRNAs, and finally the mature miRNAs produced by ZIKV. We also identified their target genes and performed functional enrichment analysis to identify the biological processes associated with these genes. Finally, we analyzed a publicly available RNA-seq data set to determine the altered expression level of the targeted genes.From ZIKV genome sequence, we identified and validated 47 putative novel miRNAs. Functional enrichment of the targeted genes demonstrates the involvement of various biological pathways regulating cellular signaling, neurological functions, cancer, and fetal development. The expression analysis of these genes showed that ZIKV-produced miRNAs downregulate the key genes involved in these pathways, which in turn may lead to impaired brain development.Our finding proposes novel ZIKV miRNAs and their targets, which upon experimental validation could help developing new therapeutics to combat ZIKV infection and minimize ZIKV-mediated pathologies.

    View details for DOI 10.1002/jmv.25505

    View details for Web of Science ID 000481450500002

    View details for PubMedID 31095749

  • Comparative Profiling of Ubiquitin Proteasome System Interplay with Influenza A Virus PB2 Polymerase Protein Recapitulating Virus Evolution in Humans MSPHERE Biquand, E., Poirson, J., Karim, M., Declercq, M., Malausse, N., Cassonnet, P., Barbezange, C., Straub, M., Jones, L., Munier, S., Naffakh, N., van der Werf, S., Jacob, Y., Masson, M., Demeret, C. 2017; 2 (6)