All Publications


  • MARCH8 Ubiquitinates the Hepatitis C Virus Nonstructural 2 Protein and Mediates Viral Envelopment. Cell reports Kumar, S. n., Barouch-Bentov, R. n., Xiao, F. n., Schor, S. n., Pu, S. n., Biquand, E. n., Lu, A. n., Lindenbach, B. D., Jacob, Y. n., Demeret, C. n., Einav, S. n. 2019; 26 (7): 1800–1814.e5

    Abstract

    The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design.

    View details for PubMedID 30759391

  • Viral journeys on the intracellular highways. Cellular and molecular life sciences : CMLS Robinson, M., Schor, S., Barouch-Bentov, R., Einav, S. 2018

    Abstract

    Viruses are obligate intracellular pathogens that are dependent on cellular machineries for their replication. Recent technological breakthroughs have facilitated reliable identification of host factors required for viral infections and better characterization of the virus-host interplay. While these studies have revealed cellular machineries that are uniquely required by individual viruses, accumulating data also indicate the presence of broadly required mechanisms. Among these overlapping cellular functions are components of intracellular membrane trafficking pathways. Here, we review recent discoveries focused on how viruses exploit intracellular membrane trafficking pathways to promote various stages of their life cycle, with an emphasis on cellular factors that are usurped by a broad range of viruses. We describe broadly required components of the endocytic and secretory pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Identification of such overlapping host functions offers new opportunities to develop broad-spectrum host-targeted antiviral strategies.

    View details for PubMedID 30043139

  • Feasibility and biological rationale of repurposing sunitinib and erlotinib for dengue treatment. Antiviral research Pu, S. Y., Xiao, F. n., Schor, S. n., Bekerman, E. n., Zanini, F. n., Barouch-Bentov, R. n., Nagamine, C. M., Einav, S. n. 2018; 155: 67–75

    Abstract

    There is an urgent need for strategies to combat dengue virus (DENV) infection; a major global threat. We reported that the cellular kinases AAK1 and GAK regulate intracellular trafficking of multiple viruses and that sunitinib and erlotinib, approved anticancer drugs with potent activity against these kinases, protect DENV-infected mice from mortality. Nevertheless, further characterization of the therapeutic potential and underlying mechanism of this approach is required prior to clinical evaluation. Here, we demonstrate that sunitinib/erlotinib combination achieves sustained suppression of systemic infection at approved dose in DENV-infected IFN-α/β and IFN-γ receptor-deficient mice. Nevertheless, treatment with these blood-brain barrier impermeable drugs delays, yet does not prevent, late-onset paralysis; a common manifestation in this immunodeficient mouse model but not in humans. Sunitinib and erlotinib treatment also demonstrates efficacy in human primary monocyte-derived dendritic cells. Additionally, DENV infection induces expression of AAK1 transcripts, but not GAK, via single-cell transcriptomics, and these kinases are important molecular targets underlying the anti-DENV effect of sunitinib and erlotinib. Lastly, sunitinib/erlotinib combination alters inflammatory cytokine responses in DENV-infected mice. These findings support feasibility of repurposing sunitinib/erlotinib combination as a host-targeted antiviral approach and contribute to understanding its mechanism of antiviral action.

    View details for PubMedID 29753658

  • Erratum for Barouch-Bentov et al., "Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment". mBio Barouch-Bentov, R. n., Neveu, G. n., Xiao, F. n., Beer, M. n., Bekerman, E. n., Schor, S. n., Campbell, J. n., Boonyaratanakornkit, J. n., Lindenbach, B. n., Lu, A. n., Jacob, Y. n., Einav, S. n. 2018; 9 (1)

    View details for PubMedID 29317514

  • Repurposing of Kinase Inhibitors as Broad-Spectrum Antiviral Drugs. DNA and cell biology Schor, S. n., Einav, S. n. 2018; 37 (2): 63–69

    Abstract

    The high cost of drug development and the narrow spectrum of coverage typically provided by direct-acting antivirals limit the scalability of this antiviral approach. This review summarizes progress and challenges in the repurposing of approved kinase inhibitors as host-targeted broad-spectrum antiviral therapies.

    View details for PubMedID 29148875

  • Optimization of Isothiazolo[4,3- b]pyridine-Based Inhibitors of Cyclin G Associated Kinase (GAK) with Broad-Spectrum Antiviral Activity. Journal of medicinal chemistry Pu, S. Y., Wouters, R. n., Schor, S. n., Rozenski, J. n., Barouch-Bentov, R. n., Prugar, L. I., O'Brien, C. M., Brannan, J. M., Dye, J. M., Herdewijn, P. n., De Jonghe, S. n., Einav, S. n. 2018

    Abstract

    There is an urgent need for strategies to combat dengue and other emerging viral infections. We reported that cyclin G-associated kinase (GAK), a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, regulates intracellular trafficking of multiple unrelated RNA viruses during early and late stages of the viral lifecycle. We also reported the discovery of potent, selective GAK inhibitors based on an isothiazolo[4,3- b]pyridine scaffold, albeit with moderate antiviral activity. Here, we describe our efforts leading to the discovery of novel isothiazolo[4,3- b]pyridines that maintain high GAK affinity and selectivity. These compounds demonstrate improved in vitro activity against dengue virus, including in human primary dendritic cells, and efficacy against the unrelated Ebola and chikungunya viruses. Moreover, inhibition of GAK activity was validated as an important mechanism of antiviral action of these compounds. These findings demonstrate the potential utility of a GAK-targeted broad-spectrum approach for combating currently untreatable emerging viral infections.

    View details for PubMedID 29953812

  • Combating Intracellular Pathogens with Repurposed Host-Targeted Drugs. ACS infectious diseases Schor, S. n., Einav, S. n. 2018; 4 (2): 88–92

    Abstract

    There is a large, global unmet need for the development of countermeasures to combat intracellular pathogens. The development of novel antimicrobials is expensive and slow and typically focuses on selective inhibition of proteins encoded by a single pathogen, thereby providing a narrow spectrum of coverage. The repurposing of approved drugs targeting host functions required for microbial infections represents a promising alternative. This review summarizes progress and challenges in the repurposing of approved drugs as host-targeted broad-spectrum agents for the treatment of intracellular pathogens. These strategies include targeting both cellular factors required for infection by various viruses, intracellular bacteria, and/or protozoa as well as factors that modulate the host immune response to these microbial infections. The repurposed approach offers complementary means to develop therapeutics against existing and emerging intracellular microbial threats.

    View details for PubMedID 29298032

  • Interactions between the Hepatitis C Virus Nonstructural 2 Protein and Host Adaptor Proteins 1 and 4 Orchestrate Virus Release. mBio Xiao, F. n., Wang, S. n., Barouch-Bentov, R. n., Neveu, G. n., Pu, S. n., Beer, M. n., Schor, S. n., Kumar, S. n., Nicolaescu, V. n., Lindenbach, B. D., Randall, G. n., Einav, S. n. 2018; 9 (2)

    Abstract

    Hepatitis C virus (HCV) spreads via secreted cell-free particles or direct cell-to-cell transmission. Yet, virus-host determinants governing differential intracellular trafficking of cell-free- and cell-to-cell-transmitted virus remain unknown. The host adaptor proteins (APs) AP-1A, AP-1B, and AP-4 traffic in post-Golgi compartments, and the latter two are implicated in basolateral sorting. We reported that AP-1A mediates HCV trafficking during release, whereas the endocytic adaptor AP-2 mediates entry and assembly. We demonstrated that the host kinases AAK1 and GAK regulate HCV infection by controlling these clathrin-associated APs. Here, we sought to define the roles of AP-4, a clathrin-independent adaptor; AP-1A; and AP-1B in HCV infection. We screened for interactions between HCV proteins and the μ subunits of AP-1A, AP-1B, and AP-4 by mammalian cell-based protein fragment complementation assays. The nonstructural 2 (NS2) protein emerged as an interactor of these adaptors in this screening and by coimmunoprecipitations in HCV-infected cells. Two previously unrecognized dileucine-based motifs in the NS2 C terminus mediated AP binding and HCV release. Infectivity and coculture assays demonstrated that while all three adaptors mediate HCV release and cell-free spread, AP-1B and AP-4, but not AP-1A, mediate cell-to-cell spread. Live-cell imaging revealed HCV cotrafficking with AP-1A, AP-1B, and AP-4 and that AP-4 mediates HCV trafficking in a post-Golgi compartment. Lastly, HCV cell-to-cell spread was regulated by AAK1 and GAK and thus susceptible to treatment with AAK1 and GAK inhibitors. These data provide a mechanistic understanding of HCV trafficking in distinct release pathways and reveal a requirement for APs in cell-to-cell viral spread.IMPORTANCE HCV spreads via cell-free infection or cell-to-cell contact that shields it from antibody neutralization, thereby facilitating viral persistence. Yet, factors governing this differential sorting remain unknown. By integrating proteomic, RNA interference, genetic, live-cell imaging, and pharmacological approaches, we uncover differential coopting of host adaptor proteins (APs) to mediate HCV traffic at distinct late steps of the viral life cycle. We reported that AP-1A and AP-2 mediate HCV trafficking during release and assembly, respectively. Here, we demonstrate that dileucine motifs in the NS2 protein mediate AP-1A, AP-1B, and AP-4 binding and cell-free virus release. Moreover, we reveal that AP-4, an adaptor not previously implicated in viral infections, mediates cell-to-cell spread and HCV trafficking. Lastly, we demonstrate cell-to-cell spread regulation by AAK1 and GAK, host kinases controlling APs, and susceptibility to their inhibitors. This study provides mechanistic insights into virus-host determinants that facilitate HCV trafficking, with potential implications for pathogenesis and antiviral agent design.

    View details for PubMedID 29535204

    View details for PubMedCentralID PMC5850324

  • Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment (vol 47, e01456-16, 2016) MBIO Barouch-Bentov, R., Neveu, G., Xiao, F., Beer, M., Bekerman, E., Schor, S., Campbell, J., Boonyaratanakornkit, J., Lindenbach, B., Lu, A., Jacob, Y., Einav, S. 2018; 9 (1)
  • Repurposing of Kinase Inhibitors as Broad-Spectrum Antiviral Drugs. DNA Cell Biol. Schor, S., Einav, S. 2017: 63–69

    Abstract

    The high cost of drug development and the narrow spectrum of coverage typically provided by direct-acting antivirals limit the scalability of this antiviral approach. This review summarizes progress and challenges in the repurposing of approved kinase inhibitors as host-targeted broad-spectrum antiviral therapies.

    View details for DOI 10.1089/dna.2017.4033

    View details for PubMedCentralID PMC5804095

  • Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment. mBio Barouch-Bentov, R., Neveu, G., Xiao, F., Beer, M., Bekerman, E., Schor, S., Campbell, J., Boonyaratanakornkit, J., Lindenbach, B., Lu, A., Jacob, Y., Einav, S. 2016; 7 (6)

    Abstract

    Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.Viruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies.

    View details for DOI 10.1128/mBio.01456-16

    View details for PubMedID 27803188

    View details for PubMedCentralID PMC5090039

  • Directed Evolution of Gloeobacter violaceus Rhodopsin Spectral Properties. Journal of molecular biology Engqvist, M. K., McIsaac, R. S., Dollinger, P., Flytzanis, N. C., Abrams, M., Schor, S., Arnold, F. H. 2015; 427 (1): 205-220

    Abstract

    Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to ±80nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton-pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurred blue and red shifts of 42nm and 22nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty eight of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the WT (wild-type) protein.

    View details for DOI 10.1016/j.jmb.2014.06.015

    View details for PubMedID 24979679

  • Identification, in Vitro Activity and Mode of Action of Phosphoinositide-Dependent-1 Kinase Inhibitors as Antifungal Molecules ACS CHEMICAL BIOLOGY Baxter, B. K., Didone, L., Ogu, D., Schor, S., Krysan, D. J. 2011; 6 (5): 502-510

    Abstract

    Although protein kinases have recently emerged as important drug targets, the anti-infective potential of protein kinase inhibitors has not been developed extensively. We identified the mammalian PDK1 inhibitor KP-372-1 as a potent antifungal molecule with activity against yeast and fungal biofilms using a screening strategy for protein kinase inhibitors that block the cell wall stress response in yeast. Genetic and biochemical studies indicate that KP-372-1 inhibits fungal PDK1 orthologs (Pkh kinases) as part of its mode of action and support a role for Pkh kinases in eisosome assembly. Two other structurally distinct molecules that inhibit PDK1, OSU-03012 and UCN-01, also have antifungal activity. Taken together, these data indicate that fungal PDK1 orthologs are promising targets for new antifungal drug development.

    View details for DOI 10.1021/cb100399x

    View details for Web of Science ID 000290747200015

    View details for PubMedID 21294551

    View details for PubMedCentralID PMC3098953