Shirit Einav

All Publications

• Replication Capacity and Susceptibility of Nirmatrelvir-Resistant Mutants to Next-Generation Mpro Inhibitors in a SARS-CoV-2 Replicon System. Antiviral research Lo, C., Kariv, O., Hao, C., Gammeltoft, K. A., Bukh, J., Gottwein, J., Westberg, M., Lin, M. Z., Einav, S. 2024: 106022

  Abstract

  There is an ongoing need to expand the anti-SARS-CoV-2 armamentarium to include agents capable of suppressing replication of drug-resistant mutants emerging during monotherapy with approved direct-acting antivirals. Using a subgenomic SARS-CoV-2 replicon system, we studied the RNA replication capacity of nirmatrelvir (NTV)-resistant mutants and their susceptibility to next-generation Mpro inhibitors, including ibuzatrelvir (ITV), ensitrelvir (ETV), and ML2006a4. Our findings revealed that E166V Mpro mutants reduced viral RNA replication, whereas other Mpro mutations retained or increased the replication capacity, suggesting the potential of the latter to dominate under NTV selective pressure. Except for having an advantage against E166A mutants, ITV largely showed the same mutational sensitivity as NTV. ETV was more effective than NTV against E166V mutants but less effective against S144A, E166A, and L167F mutants. ML2006a4 demonstrated the most effective suppression across most mutants (S144A, E166V, S144A+L50F, E166A/V+L50F, L167F+L50F, and E166A+L167F+L50F). Thus, ML2006a4 represents an attractive investigational candidate against clinically relevant NTV-resistant SARS-CoV-2 mutants.

  View details for DOI 10.1016/j.antiviral.2024.106022

  View details for PubMedID 39424074

• Synthesis of 3-heteroaryl-pyrrolo[2,3-b]pyridines as potent inhibitors of AP-2-associated protein kinase 1 (AAK1) with antiviral activity. European journal of medicinal chemistry Ravi, N. P., Van Eynde, W., Karim, M., Nhu Tran, D. H., Agrawal, A., Schols, D., Voet, A., Einav, S., Dehaen, W., De Jonghe, S. 2024; 280: 116967

  Abstract

  Inhibition of AP-2-associated protein kinase 1 (AAK1) has been shown to be a promising avenue for the development of broad-spectrum antiviral agents. On a previously described AAK1 inhibitor based on a pyrrolo[2,3-b]pyridine scaffold, the concept of isosterism was applied, by replacing a carboxamide linker by various five-membered heterocycles. It led to the discovery of a novel series of AAK1 inhibitors with IC50 values in the low nM range, that also displayed antiviral activity against the dengue virus and Venezuelan equine encephalitis virus.

  View details for DOI 10.1016/j.ejmech.2024.116967

  View details for PubMedID 39427517

• Back-Pocket Optimization of 2-Aminopyrimidine-Based Macrocycles Leads to Potent EPHA2/GAK Kinase Inhibitors. Journal of medicinal chemistry Gerninghaus, J., Zhubi, R., Krämer, A., Karim, M., Tran, D. H., Joerger, A. C., Schreiber, C., Berger, L. M., Berger, B. T., Ehret, T. A., Elson, L., Lenz, C., Saxena, K., Müller, S., Einav, S., Knapp, S., Hanke, T. 2024

  Abstract

  Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here we have optimized 2-aminopyrimidine-based macrocycles to use these compounds as chemical tools for the ephrin kinase family. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55, a potent inhibitor of EPHA2/A4 and GAK. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/A4 and GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells. However, further investigations are needed to determine whether this was a compound-specific effect or target-related.

  View details for DOI 10.1021/acs.jmedchem.4c00411

  View details for PubMedID 39028937

• Numb-associated kinases regulate sandfly-borne Toscana virus entry. Emerging microbes & infections Moalem, Y., Katz, R., Subramaniam, A. G., Malis, Y., Yaffe, Y., Borenstein-Auerbach, N., Tadmor, K., Raved, R., Maoz, B. M., Yoo, J. S., Lustig, Y., Luxenburg, C., Perlson, E., Einav, S., Sklan, E. H. 2024: 2382237

  Abstract

  Sandfly-borne Toscana virus (TOSV) is an enveloped tri-segmented negative single-strand RNA Phlebovirus. It is an emerging virus predominantly endemic in southwestern Europe and Northern Africa. Although TOSV infection is typically asymptomatic or results in mild febrile disease, it is neurovirulent and ranks among the three most common causes of summer meningitis in certain regions. Despite this clinical significance, our understanding of the molecular aspects and host factors regulating phlebovirus infection is limited.This study characterized the early steps of TOSV infection. Our findings reveal that two members of the Numb-associated kinases family of Ser/Thr kinases, namely adaptor-associated kinase 1 (AAK1) and cyclin G-associated kinase (GAK), play a role in regulating the early stages of TOSV entry. FDA-approved inhibitors targeting these kinases demonstrated significant inhibition of TOSV infection. This study suggests that AAK1 and GAK represent druggable targets for inhibiting TOSV infection and, potentially, related Phleboviruses.

  View details for DOI 10.1080/22221751.2024.2382237

  View details for PubMedID 39017647

• PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2. bioRxiv : the preprint server for biology Karim, M., Mishra, M., Lo, C. W., Saul, S., Cagirici, H. B., Tran, D. H., Agrawal, A., Ghita, L., Ojha, A., East, M. P., Gammeltoft, K. A., Sahoo, M. K., Johnson, G. L., Das, S., Jochmans, D., Cohen, C. A., Gottwein, J., Dye, J., Neff, N., Pinsky, B. A., Laitinen, T., Pantsar, T., Poso, A., Zanini, F., Jonghe, S. D., Asquith, C. R., Einav, S. 2024

  Abstract

  In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2C's roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

  View details for DOI 10.1101/2024.04.15.589676

  View details for PubMedID 38659941

  View details for PubMedCentralID PMC11042293

• An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations. Science translational medicine Westberg, M., Su, Y., Zou, X., Huang, P., Rustagi, A., Garhyan, J., Patel, P. B., Fernandez, D., Wu, Y., Hao, C., Lo, C. W., Karim, M., Ning, L., Beck, A., Saenkham-Huntsinger, P., Tat, V., Drelich, A., Peng, B. H., Einav, S., Tseng, C. K., Blish, C., Lin, M. Z. 2024; 16 (738): eadi0979

  Abstract

  Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

  View details for DOI 10.1126/scitranslmed.adi0979

  View details for PubMedID 38478629

• Chemical inactivation strategies for SARS-CoV-2-infected cells and organoids. STAR protocols Karim, M., Pohane, A. A., Lo, C. W., Einav, S., Garhyan, J. 2024; 5 (1): 102906

  Abstract

  Infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, conducted in high-containment laboratories, requires transferring samples to lower containment labs for downstream applications, mandating sample inactivation. Here, we present a stepwise protocol for chemical inactivation of SARS-CoV-2 virus in culture supernatants or within infected cells and organoids, using eight chemical reagents validated via plaque assays. Additionally, we describe steps for troubleshooting virus inactivation, titer calculation, and log reduction. This protocol offers valuable resources for the COVID-19 research community, providing essential tools to advance research on this virus.

  View details for DOI 10.1016/j.xpro.2024.102906

  View details for PubMedID 38401122

• Back-pocket optimization of 2-aminopyrimidine-based macrocycles leads to potent dual EPHA2/GAK kinase inhibitors with antiviral activity. bioRxiv : the preprint server for biology Gerninghaus, J., Zhubi, R., Krämer, A., Karim, M., Tran, D. H., Joerger, A. C., Schreiber, C., Berger, L. M., Berger, B. T., Ehret, T. A., Elson, L., Lenz, C., Saxena, K., Müller, S., Einav, S., Knapp, S., Hanke, T. 2024

  Abstract

  Macrocyclization of acyclic compounds is a powerful strategy for improving inhibitor potency and selectivity. Here, we developed a 2-aminopyrimidine-based macrocyclic dual EPHA2/GAK kinase inhibitor as a chemical tool to study the role of these two kinases in viral entry and assembly. Starting with a promiscuous macrocyclic inhibitor, 6, we performed a structure-guided activity relationship and selectivity study using a panel of over 100 kinases. The crystal structure of EPHA2 in complex with the developed macrocycle 23 provided a basis for further optimization by specifically targeting the back pocket, resulting in compound 55 as a potent dual EPHA2/GAK inhibitor. Subsequent front-pocket derivatization resulted in an interesting in cellulo selectivity profile, favoring EPHA4 over the other ephrin receptor kinase family members. The dual EPHA2/GAK inhibitor 55 prevented dengue virus infection of Huh7 liver cells, mainly via its EPHA2 activity, and is therefore a promising candidate for further optimization of its activity against dengue virus.

  View details for DOI 10.1101/2024.02.18.580805

  View details for PubMedID 38405908

  View details for PubMedCentralID PMC10888910

• Systems immunology of transcriptional responses to viral infection identifies conserved antiviral pathways across macaques and humans. Cell reports Ratnasiri, K., Zheng, H., Toh, J., Yao, Z., Duran, V., Donato, M., Roederer, M., Kamath, M., Todd, J. M., Gagne, M., Foulds, K. E., Francica, J. R., Corbett, K. S., Douek, D. C., Seder, R. A., Einav, S., Blish, C. A., Khatri, P. 2024; 43 (2): 113706

  Abstract

  Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1-3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness.

  View details for DOI 10.1016/j.celrep.2024.113706

  View details for PubMedID 38294906

• Global and cell type-specific immunological hallmarks of severe dengue progression identified via a systems immunology approach. Nature immunology Ghita, L., Yao, Z., Xie, Y., Duran, V., Cagirici, H. B., Samir, J., Osman, I., Rebellón-Sánchez, D. E., Agudelo-Rojas, O. L., Sanz, A. M., Sahoo, M. K., Robinson, M. L., Gelvez-Ramirez, R. M., Bueno, N., Luciani, F., Pinsky, B. A., Montoya, J. G., Estupiñan-Cardenas, M. I., Villar-Centeno, L. A., Rojas-Garrido, E. M., Rosso, F., Quake, S. R., Zanini, F., Einav, S. 2023

  Abstract

  Severe dengue (SD) is a major cause of morbidity and mortality. To define dengue virus (DENV) target cells and immunological hallmarks of SD progression in children's blood, we integrated two single-cell approaches capturing cellular and viral elements: virus-inclusive single-cell RNA sequencing (viscRNA-Seq 2) and targeted proteomics with secretome analysis and functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion as well as cell-cell communications point towards increased immune cell migration and inflammation in SD progressors. Concurrently, antigen-presenting cells from SD progressors demonstrate intact uptake yet impaired interferon response and antigen processing and presentation signatures, which are partly modulated by DENV. Increased activation, regulation and exhaustion of effector responses and expansion of HLA-DR-expressing adaptive-like NK cells also characterize SD progressors. These findings reveal DENV target cells in human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.

  View details for DOI 10.1038/s41590-023-01654-3

  View details for PubMedID 37872316

  View details for PubMedCentralID 3651993

• Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses. PLoS pathogens Lubow, J., Levoir, L. M., Ralph, D. K., Belmont, L., Contreras, M., Cartwright-Acar, C. H., Kikawa, C., Kannan, S., Davidson, E., Duran, V., Rebellon-Sanchez, D. E., Sanz, A. M., Rosso, F., Doranz, B. J., Einav, S., Matsen Iv, F. A., Goo, L. 2023; 19 (10): e1011722

  Abstract

  Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasures that avoid enhancement of infection associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following repeated DENV infections. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

  View details for DOI 10.1371/journal.ppat.1011722

  View details for PubMedID 37812640

• Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects. The Journal of clinical investigation Saul, S., Karim, M., Ghita, L., Huang, P. T., Chiu, W., Durán, V., Lo, C. W., Kumar, S., Bhalla, N., Leyssen, P., Alem, F., Boghdeh, N. A., Tran, D. H., Cohen, C. A., Brown, J. A., Huie, K. E., Tindle, C., Sibai, M., Ye, C., Khalil, A. M., Chiem, K., Martinez-Sobrido, L., Dye, J. M., Pinsky, B. A., Ghosh, P., Das, S., Solow-Cordero, D. E., Jin, J., Wikswo, J. P., Jochmans, D., Neyts, J., De Jonghe, S., Narayanan, A., Einav, S. 2023

  Abstract

  Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

  View details for DOI 10.1172/JCI169510

  View details for PubMedID 37581931

• Preparing for the next viral threat with broad-spectrum antivirals. The Journal of clinical investigation Karim, M., Lo, C. W., Einav, S. 2023; 133 (11)

  Abstract

  There is a large global unmet need for the development of countermeasures to combat hundreds of viruses known to cause human disease and for the establishment of a therapeutic portfolio for future pandemic preparedness. Most approved antiviral therapeutics target proteins encoded by a single virus, providing a narrow spectrum of coverage. This, combined with the slow pace and high cost of drug development, limits the scalability of this direct-acting antiviral (DAA) approach. Here, we summarize progress and challenges in the development of broad-spectrum antivirals that target either viral elements (proteins, genome structures, and lipid envelopes) or cellular proviral factors co-opted by multiple viruses via newly discovered compounds or repurposing of approved drugs. These strategies offer new means for developing therapeutics against both existing and emerging viral threats that complement DAAs.

  View details for DOI 10.1172/JCI170236

  View details for PubMedID 37259914

• Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics. Science advances Robinson, M. L., Glass, D. R., Duran, V., Agudelo Rojas, O. L., Sanz, A. M., Consuegra, M., Sahoo, M. K., Hartmann, F. J., Bosse, M., Gelvez, R. M., Bueno, N., Pinsky, B. A., Montoya, J. G., Maecker, H., Estupiñan Cardenas, M. I., Villar Centeno, L. A., Garrido, E. M., Rosso, F., Bendall, S. C., Einav, S. 2023; 9 (12): eade7702

  Abstract

  Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

  View details for DOI 10.1126/sciadv.ade7702

  View details for PubMedID 36961888

• Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX. PLoS pathogens Shirasaki, T., Feng, H., Duyvesteyn, H. M., Fusco, W. G., McKnight, K. L., Xie, L., Boyce, M., Kumar, S., Barouch-Bentov, R., Gonzalez-Lopez, O., McNamara, R., Wang, L., Hertel-Wulff, A., Chen, X., Einav, S., Duncan, J. A., Kapustina, M., Fry, E. E., Stuart, D. I., Lemon, S. M. 2022; 18 (8): e1010543

  Abstract

  Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.

  View details for DOI 10.1371/journal.ppat.1010543

  View details for PubMedID 35969644

• Numb-associated kinases are required for SARS-CoV-2 infection and are cellular targets for antiviral strategies. Antiviral research Karim, M., Saul, S., Ghita, L., Sahoo, M. K., Ye, C., Bhalla, N., Lo, C. W., Jin, J., Park, J., Martinez-Gualda, B., East, M. P., Johnson, G. L., Pinsky, B. A., Martinez-Sobrido, L., Asquith, C. R., Narayanan, A., De Jonghe, S., Einav, S. 2022: 105367

  Abstract

  The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose serious threats to global health. We previously reported that AAK1, BIKE and GAK, members of the Numb-associated kinase family, control intracellular trafficking of multiple RNA viruses during viral entry and assembly/egress. Here, using both genetic and pharmacological approaches, we probe the functional relevance of NAKs for SARS-CoV-2 infection. siRNA-mediated depletion of AAK1, BIKE, GAK, and STK16, the fourth member of the NAK family, suppressed SARS-CoV-2 infection in human lung epithelial cells. Both known and novel small molecules with potent AAK1/BIKE, GAK or STK16 activity suppressed SARS-CoV-2 infection. Moreover, combination treatment with the approved anti-cancer drugs, sunitinib and erlotinib, with potent anti-AAK1/BIKE and GAK activity, respectively, demonstrated synergistic effect against SARS-CoV-2 infection in vitro. Time-of-addition experiments revealed that pharmacological inhibition of AAK1 and BIKE suppressed viral entry as well as late stages of the SARS-CoV-2 life cycle. Lastly, suppression of NAKs expression by siRNAs inhibited entry of both wild type and SARS-CoV-2 pseudovirus. These findings provide insight into the roles of NAKs in SARS-CoV-2 infection and establish a proof-of-principle that pharmacological inhibition of NAKs can be potentially used as a host-targeted approach to treat SARS-CoV-2 with potential implications to other coronaviruses.

  View details for DOI 10.1016/j.antiviral.2022.105367

  View details for PubMedID 35738348

• The cargo adaptor protein CLINT1 is phosphorylated by the Numb-associated kinase BIKE and mediates dengue virus infection. The Journal of biological chemistry Schor, S., Pu, S., Nicolaescu, V., Azari, S., Koivomagi, M., Karim, M., Cassonnet, P., Saul, S., Neveu, G., Yueh, A., Demeret, C., Skotheim, J. M., Jacob, Y., Randall, G., Einav, S. 2022: 101956

  Abstract

  The signaling pathways and cellular functions regulated by the four Numb-associated kinases (NAKs) are largely unknown. We previously reported that AAK1 and GAK control intracellular trafficking of RNA viruses, and recently revealed a requirement for BIKE in early and late stages of dengue virus (DENV) infection. However, the downstream targets phosphorylated by BIKE in this process have not yet been identified. Here, to identify BIKE substrates, we conducted a barcode fusion genetics-yeast two-hybrid screen and retrieved publicly available data generated via affinity-purification mass spectrometry. We subsequently validated 19 of 47 putative BIKE interactors using mammalian cell-based protein-protein interaction assays. We found that CLINT1, a cargo-specific adaptor implicated in bidirectional Golgi-to-endosome trafficking, emerged as a predominant hit in both screens. Our experiments indicated that BIKE catalyzes phosphorylation of a threonine 294 (T294) CLINT1 residue both in vitro and in cell culture. Our findings revealed that CLINT1 phosphorylation mediates its binding to the DENV nonstructural 3 protein and subsequently promotes DENV assembly and egress. In addition, using live-cell imaging we revealed that CLINT1 cotraffics with DENV particles and is involved in mediating BIKE's role in DENV infection. Finally, our data suggest that additional cellular BIKE interactors implicated in the host immune and stress responses and the ubiquitin proteasome system might also be candidate phosphorylation substrates of BIKE. In conclusion, these findings reveal cellular substrates and pathways regulated by the understudied NAK enzyme BIKE, a mechanism for CLINT1 regulation, and control of DENV infection via BIKE signaling, with potential implications for cell biology, virology, and host-targeted antiviral design.

  View details for DOI 10.1016/j.jbc.2022.101956

  View details for PubMedID 35452674

• An 8-gene machine learning model improves clinical prediction of severe dengue progression. Genome medicine Liu, Y. E., Saul, S., Rao, A. M., Robinson, M. L., Agudelo Rojas, O. L., Sanz, A. M., Verghese, M., Solis, D., Sibai, M., Huang, C. H., Sahoo, M. K., Gelvez, R. M., Bueno, N., Estupinan Cardenas, M. I., Villar Centeno, L. A., Rojas Garrido, E. M., Rosso, F., Donato, M., Pinsky, B. A., Einav, S., Khatri, P. 2022; 14 (1): 33

  Abstract

  BACKGROUND: Each year 3-6 million people develop life-threatening severe dengue (SD). Clinical warning signs for SD manifest late in the disease course and are nonspecific, leading to missed cases and excess hospital burden. Better SD prognostics are urgently needed.METHODS: We integrated 11 public datasets profiling the blood transcriptome of 365 dengue patients of all ages and from seven countries, encompassing biological, clinical, and technical heterogeneity. We performed an iterative multi-cohort analysis to identify differentially expressed genes (DEGs) between non-severe patients and SD progressors. Using only these DEGs, we trained an XGBoost machine learning model on public data to predict progression to SD. All model parameters were "locked" prior to validation in an independent, prospectively enrolled cohort of 377 dengue patients in Colombia. We measured expression of the DEGs in whole blood samples collected upon presentation, prior to SD progression. We then compared the accuracy of the locked XGBoost model and clinical warning signs in predicting SD.RESULTS: We identified eight SD-associated DEGs in the public datasets and built an 8-gene XGBoost model that accurately predicted SD progression in the independent validation cohort with 86.4% (95% CI 68.2-100) sensitivity and 79.7% (95% CI 75.5-83.9) specificity. Given the 5.8% proportion of SD cases in this cohort, the 8-gene model had a positive and negative predictive value (PPV and NPV) of 20.9% (95% CI 16.7-25.6) and 99.0% (95% CI 97.7-100.0), respectively. Compared to clinical warning signs at presentation, which had 77.3% (95% CI 58.3-94.1) sensitivity and 39.7% (95% CI 34.7-44.9) specificity, the 8-gene model led to an 80% reduction in the number needed to predict (NNP) from 25.4 to 5.0. Importantly, the 8-gene model accurately predicted subsequent SD in the first three days post-fever onset and up to three days prior to SD progression.CONCLUSIONS: The 8-gene XGBoost model, trained on heterogeneous public datasets, accurately predicted progression to SD in a large, independent, prospective cohort, including during the early febrile stage when SD prediction remains clinically difficult. The model has potential to be translated to a point-of-care prognostic assay to reduce dengue morbidity and mortality without overwhelming limited healthcare resources.

  View details for DOI 10.1186/s13073-022-01034-w

  View details for PubMedID 35346346

• Synthesis and evaluation of 3-alkynyl-5-aryl-7-aza-indoles as broad-spectrum antiviral agents. Frontiers in chemistry Martinez-Gualda, B., Graus, M., Camps, A., Vanhulle, E., Saul, S., Azari, S., Nhu Tran, D. H., Vangeel, L., Chiu, W., Neyts, J., Schols, D., Einav, S., Vermeire, K., De Jonghe, S. 2022; 10: 1058229

  Abstract

  RNA viral infections, including those caused by respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Venezuelan Equine encephalitis virus (VEEV), pose a major global health challenge. Here, we report the synthesis and screening of a series of pyrrolo[2,3-b]pyridines targeting RSV, SARS-CoV-2 and/or VEEV. From this campaign, a series of lead compounds was generated that demonstrated antiviral activity in the low single-digit micromolar range against the various viruses and did not show cytotoxicity. These findings highlight the potential of 3-alkynyl-5-aryl-7-aza-indoles as a promising chemotype for the development of broad-spectrum antiviral agents.

  View details for DOI 10.3389/fchem.2022.1058229

  View details for PubMedID 36385995

• Optimization of 4-Anilinoquinolines as Dengue Virus Inhibitors. Molecules (Basel, Switzerland) Huang, P., Saul, S., Einav, S., Asquith, C. R. 2021; 26 (23)

  Abstract

  Emerging viral infections, including those caused by dengue virus (DENV) and Venezuelan Equine Encephalitis virus (VEEV), pose a significant global health challenge. Here, we report the preparation and screening of a series of 4-anilinoquinoline libraries targeting DENV and VEEV. This effort generated a series of lead compounds, each occupying a distinct chemical space, including 3-((6-bromoquinolin-4-yl)amino)phenol (12), 6-bromo-N-(5-fluoro-1H-indazol-6-yl)quinolin-4-amine (50) and 6-((6-bromoquinolin-4-yl)amino)isoindolin-1-one (52), with EC50 values of 0.63-0.69 M for DENV infection. These compound libraries demonstrated very limited toxicity with CC50 values greater than 10 M in almost all cases. Additionally, the lead compounds were screened for activity against VEEV and demonstrated activity in the low single-digit micromolar range, with 50 and 52 demonstrating EC50s of 2.3 M and 3.6 M, respectively. The promising results presented here highlight the potential to further refine this series in order to develop a clinical compound against DENV, VEEV, and potentially other emerging viral threats.

  View details for DOI 10.3390/molecules26237338

  View details for PubMedID 34885921

• PIKfyve: a lipid kinase target for COVID-19, cancer and neurodegenerative disorders NATURE REVIEWS DRUG DISCOVERY Huang, P., Einav, S., Asquith, C. M. 2021; 20 (10): 730

  View details for DOI 10.1038/d41573-021-00158-9

  View details for Web of Science ID 000701704700008

  View details for PubMedID 34504321

• Discovery of 3-phenyl- and 3-N-piperidinyl-isothiazolo[4,3-b]pyridines as highly potent inhibitors of cyclin G-associated kinase. European journal of medicinal chemistry Martinez-Gualda, B., Saul, S., Froeyen, M., Schols, D., Herdewijn, P., Einav, S., De Jonghe, S. 2021; 213: 113158

  Abstract

  Structural modifications at position 3 of the isothiazolo[4,3-b]pyridine scaffold afforded a new series of cyclin G-associated kinase (GAK) inhibitors. It was shown that the insertion of a carboxamide residue at position 3 of a phenyl or piperidinyl moiety generated potent GAK inhibitors with IC50 values in a low nanomolar range. This potent GAK binding affinity was rationalized by molecular modelling demonstrating that the carboxamide moiety engages in an extra hydrogen bond with GAK. Moreover, this new series of compounds was also endowed with antiviral activity against dengue virus, highlighting the potential utility of GAK as a target for the development of antiviral drugs.

  View details for DOI 10.1016/j.ejmech.2021.113158

  View details for PubMedID 33497888

• 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

• Evaluation and identification of 4-anilinoquin(az)olines as potent inhibitors of both dengue virus (DENV) and Venezuelan equine encephalitis virus (VEEV). Bioorganic & medicinal chemistry letters Saul, S., Huang, P. T., Einav, S., Asquith, C. R. 2021: 128407

  Abstract

  There is an urgent need for novel strategies for the treatment of emerging arthropod-borne viral infections, including those caused by dengue virus (DENV) and Venezuelan equine encephalitis virus (VEEV). We prepared and screened focused libraries of 4-anilinoquinolines and 4-anilinoquinazolines for antiviral activity and identified three potent compounds. N-(2,5-dimethoxyphenyl)-6-(trifluoromethyl)quinolin-4-amine (10) inhibited DENV infection with an EC50 = 0.25 µM, N-(3,4-dichlorophenyl)-6-(trifluoromethyl)quinolin-4-amine (27) demonstrated an EC50 = 0.50 µM against VEEV, and an advanced lead, N-(3-ethynyl-4-fluorophenyl)-6,7-dimethoxyquinazolin-4-amine (54) had a potent antiviral activity (EC50 = 0.60 µM) for VEEV. These series of compounds demonstrated nearly no toxicity with CC50 values greater than 10 µM in all cases. These promising results provide a future prospective to develop a clinical compound against these emerging viral threats.

  View details for DOI 10.1016/j.bmcl.2021.128407

  View details for PubMedID 34624490

• 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

• Potent antiviral activity of novel multi-substituted 4-anilinoquin(az)olines. Bioorganic & medicinal chemistry letters Saul, S., Pu, S., Zuercher, W. J., Einav, S., Asquith, C. R. 2020; 30 (16): 127284

  Abstract

  Screening a series of 4-anilinoquinolines and 4-anilinoquinazolines enabled identification of potent novel inhibitors of dengue virus (DENV). Preparation of focused 4-anilinoquinoline/quinazoline scaffold arrays led to the identification of a series of high potency 6-substituted bromine and iodine derivatives. The most potent compound 6-iodo-4-((3,4,5-trimethoxyphenyl)amino)quinoline-3-carbonitrile (47) inhibited DENV infection with an EC50=79nM. Crucially, these compounds showed very limited toxicity with CC50 values >10M in almost all cases. This new promising series provides an anchor point for further development to optimize compound properties.

  View details for DOI 10.1016/j.bmcl.2020.127284

  View details for PubMedID 32631507

• Towards Predicting Progression to Severe Dengue. Trends in microbiology Robinson, M. n., Einav, S. n. 2020

  Abstract

  There is an urgent need for prognostic assays to predict progression to severe dengue infection, which is a major global threat. While the majority of symptomatic dengue patients experience an acute febrile illness, 5-20% progress to severe infection associated with significant morbidity and mortality. Early monitoring and administration of supportive care reduce mortality and clinically usable biomarkers to predict severe dengue are needed. Here, we review recent discoveries of gene sets, anti-dengue antibody properties, and inflammatory markers with potential utility as predictors of disease progression. Upon larger scale validation and development of affordable sample-to-answer technologies, some of these biomarkers may be utilized to develop the first prognostic assay for improving patient care and allocating healthcare resources more effectively in dengue endemic countries.

  View details for DOI 10.1016/j.tim.2019.12.003

  View details for PubMedID 31982232

• Old Drugs for a New Virus: Repurposed Approaches for Combating COVID-19. ACS infectious diseases Saul, S. n., Einav, S. n. 2020

  Abstract

  There is a large global unmet need for effective countermeasures to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). The development of novel antiviral drugs is expensive and too slow to meet the immediate need. The repurposing of drugs that are approved or are under advanced clinical investigation provides a cost- and time-effective therapeutic solution. This review summarizes the major repurposed approaches that have been proposed or are already being studied in clinical trials for COVID-19. Among these approaches are drugs that aim to reduce SARS-CoV-2 replication by targeting either viral enzymatic functions or cellular factors required for the viral life cycle. Drugs that modulate the host immune response to SARS-CoV-2 infection by boosting it to enhance viral clearance or by suppressing it to prevent excessive inflammation and tissue injury represent another category. Lastly, we discuss means to discover repurposed drugs and the ongoing challenges associated with the off-label use of existing drugs in the context of the COVID-19 outbreak.

  View details for DOI 10.1021/acsinfecdis.0c00343

  View details for PubMedID 32687696

  View details for PubMedCentralID PMC7416635

• Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics. eLife Durham, N. D., Agrawal, A., Waltari, E., Croote, D., Zanini, F., Fouch, M., Davidson, E., Smith, O., Carabajal, E., Pak, J. E., Doranz, B. J., Robinson, M., Sanz, A. M., Albornoz, L. L., Rosso, F., Einav, S., Quake, S. R., McCutcheon, K. M., Goo, L. 2019; 8

  Abstract

  Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified J9 and J8, two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies showed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against DENV with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested that J9 and J8 followed divergent somatic hypermutation pathways, and that a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a new epitope that can be exploited for vaccine design.

  View details for DOI 10.7554/eLife.52384

  View details for PubMedID 31820734

• Structure-activity relationship study of the pyridine moiety of isothiazolo[4,3-b]pyridines as antiviral agents targeting cyclin G-associated kinase. Bioorganic & medicinal chemistry Martinez-Gualda, B., Pu, S., Froeyen, M., Herdewijn, P., Einav, S., De Jonghe, S. 2019: 115188

  Abstract

  Previously, we reported the discovery of 3,6-disubstituted isothiazolo[4,3-b]pyridines as potent and selective cyclin G-associated kinase (GAK) inhibitors with promising antiviral activity. In this manuscript, the structure-activity relationship study was expanded to synthesis of isothiazolo[4,3-b]pyridines with modifications of the pyridine moiety. This effort led to the discovery of an isothiazolo[4,3-b]pyridine derivative with a 3,4-dimethoxyphenyl residue at position 5 that displayed low nanomolar GAK binding affinity and antiviral activity against dengue virus.

  View details for DOI 10.1016/j.bmc.2019.115188

  View details for PubMedID 31757682

• A 20-Gene Set Predictive of Progression to Severe Dengue. Cell reports Robinson, M., Sweeney, T. E., Barouch-Bentov, R., Sahoo, M. K., Kalesinskas, L., Vallania, F., Sanz, A. M., Ortiz-Lasso, E., Albornoz, L. L., Rosso, F., Montoya, J. G., Pinsky, B. A., Khatri, P., Einav, S. 2019; 26 (5): 1104

  Abstract

  There is a need to identify biomarkers predictive of severe dengue. Single-cohort transcriptomics has not yielded generalizable results or parsimonious, predictive gene sets. We analyzed blood samples of dengue patients from seven gene expression datasets (446 samples, five countries) using an integrated multi-cohort analysis framework and identified a 20-gene set that predicts progression to severe dengue. We validated the predictive power of this 20-gene set in three retrospective dengue datasets (84 samples, three countries) and a prospective Colombia cohort (34 patients), with an area under the receiver operating characteristic curve of 0.89, 100% sensitivity, and 76% specificity. The 20-gene dengue severity scores declined during the diseasecourse, suggesting an infection-triggered host response. This 20-gene set is strongly associated with the progression to severe dengue and represents a predictive signature, generalizable across ages, host genetic factors, and virus strains, with potential implications for the development of a host response-based dengue prognostic assay.

  View details for PubMedID 30699342

• A 20-Gene Set Predictive of Progression to Severe Dengue CELL REPORTS Robinson, M., Sweeney, T. E., Barouch-Bentov, R., Sahoo, M., Kalesinskas, L., Vallania, F., Maria Sanz, A., Ortiz-Lasso, E., Luis Albornoz, L., Rosso, F., Montoya, J. G., Pinsky, B. A., Khatri, P., Einav, S. 2019; 26 (5): 1104-+

  View details for DOI 10.1016/j.celrep.2019.01.033

  View details for Web of Science ID 000456952400005

• Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay. Methods in molecular biology (Clifton, N.J.) Barouch-Bentov, R. n., Jacob, Y. n., Einav, S. n. 2019; 1998: 291–304

  Abstract

  The endosomal sorting complex required for transport (ESCRT) machinery comprises five complexes that act sequentially to recruit and cluster transmembrane cargo proteins (ESCRT-0), drive membrane curving (ESCRT-I and II), catalyze fission of cargo-containing vesicles (ESCRT-III and VPS/VTA1), and disassemble and recycle the ESCRT-III complex (VPS/VTA1). Since interactions between ESCRT components and cellular or microbial proteins are typically weak, transient, and involve membrane proteins, they are often difficult to study by standard technologies. Here, we describe the utility of high-throughput protein-fragment complementation assays based on the reconstitution of a split luciferase reporter to screen for interactions between any protein and a library of ESCRT proteins in mammalian cells and provide a detailed protocol for these assays.

  View details for DOI 10.1007/978-1-4939-9492-2_21

  View details for PubMedID 31250310

• 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

• Synthesis and Structure-Activity Relationships of 3,5-Disubstituted-pyrrolo[2,3- b]pyridines as Inhibitors of Adaptor-Associated Kinase 1 with Antiviral Activity. Journal of medicinal chemistry Verdonck, S. n., Pu, S. Y., Sorrell, F. J., Elkins, J. M., Froeyen, M. n., Gao, L. J., Prugar, L. I., Dorosky, D. E., Brannan, J. M., Barouch-Bentov, R. n., Knapp, S. n., Dye, J. M., Herdewijn, P. n., Einav, S. n., De Jonghe, S. n. 2019

  Abstract

  There are currently no approved drugs for the treatment of emerging viral infections, such as dengue and Ebola. Adaptor-associated kinase 1 (AAK1) is a cellular serine-threonine protein kinase that functions as a key regulator of the clathrin-associated host adaptor proteins and regulates the intracellular trafficking of multiple unrelated RNA viruses. Moreover, AAK1 is overexpressed specifically in dengue virus-infected but not bystander cells. Because AAK1 is a promising antiviral drug target, we have embarked on an optimization campaign of a previously identified 7-azaindole analogue, yielding novel pyrrolo[2,3- b]pyridines with high AAK1 affinity. The optimized compounds demonstrate improved activity against dengue virus both in vitro and in human primary dendritic cells and the unrelated Ebola virus. These findings demonstrate that targeting cellular AAK1 may represent a promising broad-spectrum antiviral strategy.

  View details for DOI 10.1021/acs.jmedchem.9b00136

  View details for PubMedID 31136173

• Virus-inclusive single-cell RNA sequencing reveals the molecular signature of progression to severe dengue. Proceedings of the National Academy of Sciences of the United States of America Zanini, F., Robinson, M. L., Croote, D., Sahoo, M. K., Sanz, A. M., Ortiz-Lasso, E., Albornoz, L. L., Rosso, F., Montoya, J. G., Goo, L., Pinsky, B. A., Quake, S. R., Einav, S. 2018

  Abstract

  Dengue virus (DENV) infection can result in severe complications. However, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here, we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes, particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes, were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes, followed by monocytes. Bystander, non-vRNA-containing B cells also demonstrated immune activation, and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly, assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD.

  View details for PubMedID 30530648

• Cyclin G-associated kinase (GAK) affinity and antiviral activity studies of a series of 3-C-substituted isothiazolo[4,3-b]pyridines. European journal of medicinal chemistry Wouters, R., Pu, S., Froeyen, M., Lescrinier, E., Einav, S., Herdewijn, P., De Jonghe, S. 2018; 163: 256–65

  Abstract

  Cyclin G-associated kinase (GAK) is a cellular regulator of the clathrin-associated host adaptor proteins AP-1 and AP-2, which regulates intracellular trafficking of dengue virus during early and late stages of the viral lifecycle. Previously, the discovery of isothiazolo[4,3-b]pyridines as potent and selective GAK inhibitors with promising antiviral activity was reported. In this manuscript, the synthesis of isothiazolo[4,3-b]pyridines with a carbon-linked substituent at position 3 is described by the application of regioselective Suzuki and Sonogashira coupling reactions. A derivative with a 3,4-dimethoxyphenyl residue at position 3 demonstrates low nanomolar binding affinity for GAK and antiviral activity against dengue virus. These findings reveal that appropriate substitution of a phenyl moiety at position 3 of the scaffold can improve GAK binding affinity.

  View details for PubMedID 30529544

• 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

• Repurposing of Kinase Inhibitors as Broad-Spectrum Antiviral Drugs. DNA and cell biology Schor, S., Einav, S. 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 DOI 10.1089/dna.2017.4033

  View details for PubMedID 29148875

  View details for PubMedCentralID PMC5804095

• Single-cell transcriptional dynamics of flavivirus infection. eLife Zanini, F. n., Pu, S. Y., Bekerman, E. n., Einav, S. n., Quake, S. R. 2018; 7

  Abstract

  Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

  View details for PubMedID 29451494

• 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)

  View details for DOI 10.1128/mBio.02234-17

  View details for Web of Science ID 000427006300095

• 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

• 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

• Turning Up Your Nose for a Flaviviral Encephalitis Cure. Cell host & microbe Barouch-Bentov, R. n., Einav, S. n. 2018; 23 (4): 427–29

  Abstract

  siRNA approaches have demonstrated promise in treating viral infections in animal models, but poor delivery limits clinical application. In this issue of Cell Host & Microbe, Beloor et al. (2018) report that nose-to-brain delivery of viral-targeted siRNA cures mice from West Nile virus encephalitis, with potential implications for human infection.

  View details for PubMedID 29649437

• 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

• Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects JOURNAL OF CLINICAL INVESTIGATION Bekerman, E., Neveu, G., Shulla, A., Brannan, J., Pu, S., Wang, S., Xiao, F., Barouch-Bentov, R., Bakken, R. R., Mateo, R., Govero, J., Nagamine, C. M., Diamond, M. S., De Jonghe, S., Herdewijn, P., Dye, J. M., Randall, G., Einav, S. 2017; 127 (4): 1338-1352

  Abstract

  Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

  View details for DOI 10.1172/JCI89857

  View details for Web of Science ID 000398183300024

  View details for PubMedID 28240606

• 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

• Pathogen receptor discovery with a microfluidic human membrane protein array PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Glick, Y., Ben-Ari, Y., Drayman, N., Pellach, M., Neveu, G., Boonyaratanakornkit, J., Avrahami, D., Einav, S., Oppenheim, A., Gerber, D. 2016; 113 (16): 4344-4349

  Abstract

  The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein-pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism.

  View details for DOI 10.1073/pnas.1518698113

  View details for Web of Science ID 000374393800043

  View details for PubMedID 27044079

  View details for PubMedCentralID PMC4843447

• Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus PLOS BIOLOGY Plissonnier, M., Lahlali, T., Michelet, M., Lebosse, F., Cottarel, J., Beer, M., Neveu, G., Durantel, D., Bartosch, B., Accardi, R., Clement, S., Paradisi, A., Devouassoux-Shisheboran, M., Einav, S., Mehlen, P., Zoulim, F., Parent, R. 2016; 14 (3)

  Abstract

  Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.

  View details for DOI 10.1371/journal.pbio.1002421

  View details for Web of Science ID 000373038200035

  View details for PubMedCentralID PMC4816328

• Response—Applying antibiotics lessons to antivirals. Science Bekerman, E., Einav, S. 2015; 348 (6242): 1437-?

  View details for DOI 10.1126/science.348.6242.1437-b

  View details for PubMedID 26113710

• Selective Inhibitors of Cyclin G Associated Kinase (GAK) as Anti-Hepatitis C Agents JOURNAL OF MEDICINAL CHEMISTRY Kovackova, S., Chang, L., Bekerman, E., Neveu, G., Barouch-Bentov, R., Chaikuad, A., Heroven, C., Sala, M., De Jonghe, S., Knapp, S., Einav, S., Herdewijn, P. 2015; 58 (8): 3393-3410

  Abstract

  Cyclin-G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Co-crystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV lifecycle (i.e. viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer and Parkinson's disease).

  View details for DOI 10.1021/jm501759m

  View details for Web of Science ID 000353602200009

  View details for PubMedID 25822739

• Infectious disease. Combating emerging viral threats. Science Bekerman, E., Einav, S. 2015; 348 (6232): 282-283

  View details for DOI 10.1126/science.aaa3778

  View details for PubMedID 25883340

• AP-2-Associated Protein Kinase 1 and Cyclin G-Associated Kinase Regulate Hepatitis C Virus Entry and Are Potential Drug Targets JOURNAL OF VIROLOGY Neveu, G., Ziv-Av, A., Barouch-Bentov, R., Berkerman, E., Mulholland, J., Einav, S. 2015; 89 (8): 4387-4404

  Abstract

  Hepatitis C virus (HCV) enters its target cell via clathrin-mediated endocytosis. AP-2-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK) are host kinases that regulate clathrin adaptor protein (AP)-mediated trafficking in the endocytic and secretory pathways. We previously reported that AAK1 and GAK regulate HCV assembly by stimulating binding of the μ subunit of AP-2, AP2M1, to HCV core protein. We also discovered that AAK1 and GAK inhibitors, including the approved anticancer drugs sunitinib and erlotinib, could block HCV assembly. Here, we hypothesized that AAK1 and GAK regulate HCV entry independently of their effect on HCV assembly. Indeed, silencing AAK1 and GAK expression inhibited entry of pseudoparticles and cell culture grown-HCV and internalization of Dil-labeled HCV particles with no effect on HCV attachment or RNA replication. AAK1 or GAK depletion impaired epidermal growth factor (EGF)-mediated enhanced HCV entry and endocytosis of EGF receptor (EGFR), an HCV entry cofactor and erlotinib's cancer target. Moreover, either RNA interference-mediated depletion of AP2M1 or NUMB, each a substrate of AAK1 and/or GAK, or overexpression of either an AP2M1 or NUMB phosphorylation site mutant inhibited HCV entry. Last, in addition to affecting assembly, sunitinib and erlotinib inhibited HCV entry at a postbinding step, their combination was synergistic, and their antiviral effect was reversed by either AAK1 or GAK overexpression. Together, these results validate AAK1 and GAK as critical regulators of HCV entry that function in part by activating EGFR, AP2M1, and NUMB and as the molecular targets underlying the antiviral effect of sunitinib and erlotinib (in addition to EGFR), respectively.Understanding the host pathways hijacked by HCV is critical for developing host-centered anti-HCV approaches. Entry represents a potential target for antiviral strategies; however, no FDA-approved HCV entry inhibitors are currently available. We reported that two host kinases, AAK1 and GAK, regulate HCV assembly. Here, we provide evidence that AAK1 and GAK regulate HCV entry independently of their role in HCV assembly and define the mechanisms underlying AAK1- and GAK-mediated HCV entry. By regulating temporally distinct steps in the HCV life cycle, AAK1 and GAK represent "master regulators" of HCV infection and potential targets for antiviral strategies. Indeed, approved anticancer drugs that potently inhibit AAK1 or GAK inhibit HCV entry in addition to assembly. These results contribute to an understanding of the mechanisms of HCV entry and reveal attractive host targets for antiviral strategies as well as approved candidate inhibitors of these targets, with potential implications for other viruses that hijack clathrin-mediated pathways.

  View details for DOI 10.1128/JVI.02705-14

  View details for Web of Science ID 000352218400030

  View details for PubMedID 25653444

• Isothiazolo[4,3-b]pyridines as inhibitors of cyclin G associated kinase: synthesis, structure-activity relationship studies and antiviral activity MEDCHEMCOMM Li, J., Kovackova, S., Pu, S., Rozenski, J., De Jonghe, S., Einav, S., Herdewijn, P. 2015; 6 (9): 1666-1672

  Abstract

  Isothiazolo[4,3-b]pyridines are known to be endowed with potent affinity for cyclin G associated kinase (GAK). In this paper, we expanded the structure-activity relationship study by broadening the structural variety at position 3 of the isothiazolo[4,3-b]pyridine scaffold. The most potent GAK ligands (displaying Kd values of less than 100 nM) within this series carry an alkoxy group at position 3 of the central scaffold. Unfortunately, these ligands display only modest antiviral activity against the hepatitis C virus.

  View details for DOI 10.1039/c5md00229j

  View details for Web of Science ID 000360639300011

  View details for PubMedCentralID PMC4763718

• B-cell receptors expressed by lymphomas of hepatitis C virus (HCV)-infected patients rarely react with the viral proteins. Blood Ng, P. P., Kuo, C., Wang, S., Einav, S., Arcaini, L., Paulli, M., Portlock, C. S., Marcotrigiano, J., Tarr, A., Ball, J., Levy, R., Levy, S. 2014; 123 (10): 1512-1515

  Abstract

  Chronic HCV infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this comes from clinical observations of tumor regressions upon anti-viral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon anti-viral treatments. We expressed the lymphoma B-cell receptors (BCRs) as soluble IgGs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region (V) gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus.

  View details for DOI 10.1182/blood-2013-10-532895

  View details for PubMedID 24449209

• Identification and Targeting of an Interaction between a Tyrosine Motif within Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly PLOS PATHOGENS Neveu, G., Barouch-Bentov, R., Ziv-Av, A., Gerber, D., Jacob, Y., Einav, S. 2012; 8 (8)

  Abstract

  Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development.

  View details for DOI 10.1371/journal.ppat.1002845

  View details for Web of Science ID 000308558000023

  View details for PubMedID 22916011

  View details for PubMedCentralID PMC3420927

• The hepatitis C virus (HCV) NS4B RNA binding inhibitor clemizole is highly synergistic with HCV protease inhibitors. journal of infectious diseases Einav, S., Sobol, H. D., Gehrig, E., Glenn, J. S. 2010; 202 (1): 65-74

  Abstract

  We recently identified a compound, clemizole hydrochloride, that inhibits NS4B's RNA binding and hepatitis C virus (HCV) replication. Although significant, clemizole's antiviral effect is moderate (50% effective concentration of 8 microM against an HCV genotype 2a clone). We hypothesized that the combination of clemizole with other anti-HCV agents can increase the antiviral effect over that achieved with each drug alone and could also decrease the emergence of viral resistance.Luciferase reporter-linked HCV replication assays were used to study the antiviral effects of drug combinations that included clemizole. Data were analyzed using Loewe additivity and Bliss independence models for synergy, and resistance studies were performed using HCV colony formation assays.Clemizole's antiviral effect was highly synergistic with the HCV protease inhibitors SCH503034 and VX950, without toxicity. In contrast, combinations of clemizole with either interferon, ribavirin, or the nucleoside (NM283) and nonnucleoside (HCV796) HCV polymerase inhibitors were additive. Furthermore, combination of clemizole with SCH503034 decreased the frequency of drug-resistant mutants, compared with treatment with either drug alone. Finally, no cross-resistance to clemizole of SCH503034-resistant mutants (or vice versa) was observed.Clemizole can yield high-level synergy with the protease inhibitor class. Inclusion of clemizole in future anti-HCV cocktails can represent an attractive paradigm for increasing current virologic response rates.

  View details for DOI 10.1086/653080

  View details for PubMedID 20486856

  View details for PubMedCentralID PMC3008401

• The Hepatitis C Virus (HCV) NS4B RNA Binding Inhibitor Clemizole Is Highly Synergistic with HCV Protease Inhibitors Annual Meeting of the Infectious-Diseases-Society-of-America Einav, S., Dvory-Sobol, H., Gehrig, E., Glenn, J. S. OXFORD UNIV PRESS INC. 2010: 65–74

  Abstract

  We recently identified a compound, clemizole hydrochloride, that inhibits NS4B's RNA binding and hepatitis C virus (HCV) replication. Although significant, clemizole's antiviral effect is moderate (50% effective concentration of 8 microM against an HCV genotype 2a clone). We hypothesized that the combination of clemizole with other anti-HCV agents can increase the antiviral effect over that achieved with each drug alone and could also decrease the emergence of viral resistance.Luciferase reporter-linked HCV replication assays were used to study the antiviral effects of drug combinations that included clemizole. Data were analyzed using Loewe additivity and Bliss independence models for synergy, and resistance studies were performed using HCV colony formation assays.Clemizole's antiviral effect was highly synergistic with the HCV protease inhibitors SCH503034 and VX950, without toxicity. In contrast, combinations of clemizole with either interferon, ribavirin, or the nucleoside (NM283) and nonnucleoside (HCV796) HCV polymerase inhibitors were additive. Furthermore, combination of clemizole with SCH503034 decreased the frequency of drug-resistant mutants, compared with treatment with either drug alone. Finally, no cross-resistance to clemizole of SCH503034-resistant mutants (or vice versa) was observed.Clemizole can yield high-level synergy with the protease inhibitor class. Inclusion of clemizole in future anti-HCV cocktails can represent an attractive paradigm for increasing current virologic response rates.

  View details for DOI 10.1086/653080

  View details for Web of Science ID 000278322300008

  View details for PubMedCentralID PMC3008401

• A small molecule inhibits HCV replication and alters NS4B's subcellular distribution ANTIVIRAL RESEARCH Bryson, P. D., Cho, N., Einav, S., Lee, C., Tai, V., Bechtel, J., Sivaraja, M., Roberts, C., Schmitz, U., Glenn, J. S. 2010; 87 (1): 1-8

  Abstract

  Hepatitis C Virus (HCV) is a leading cause of liver disease and represents a significant public health challenge. Treatments for this disease are inadequate and improved antiviral therapies are necessary. Several such antivirals are in development, most of which target the well-characterized NS3 protease or the NS5B polymerase. In contrast, the nonstructural 4B (NS4B) protein, though essential for HCV RNA replication, has been the subject of few pharmacological studies. One of the functions ascribed to this protein is the ability to form intracellular membrane-associated foci (MAF), which are believed to be related to the sites of viral replication. Here, we report the identification of a small molecule that inhibits HCV replication and disrupts the organization of these MAF. Genetic analysis links the compound's mode of action to the NS4B gene product, and transient transfections of NS4B-GFP demonstrate that treatment with this compound can lead to the formation of novel elongated assemblies of NS4B. Furthermore, an in vitro dynamic light scattering assay provides evidence that the second amphipathic helix of NS4B may be the target of the drug. Our results demonstrate that this molecule represents a new potential class of HCV inhibitors and also provides us with a useful tool for studying the HCV life cycle.

  View details for DOI 10.1016/j.antiviral.2010.03.013

  View details for PubMedID 20363257

• 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)

  Abstract

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

  View details for DOI 10.1371/journal.ppat.1000764

  View details for PubMedID 20169186

• Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis NATURE BIOTECHNOLOGY Einav, S., Gerber, D., Bryson, P. D., Sklan, E. H., Elazar, M., Maerkl, S. J., Glenn, J. S., Quake, S. R. 2008; 26 (9): 1019-1027

  Abstract

  More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (Kd) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.

  View details for DOI 10.1038/nbt.1490

  View details for PubMedID 18758449

• The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without ha-ras co-transfection HEPATOLOGY Einav, S., Sklan, E. H., Moon, H. M., Gehrig, E., Liu, P., Hao, Y., Lowe, A. W., Glenn, J. S. 2008; 47 (3): 827-835

  Abstract

  Hepatitis C virus (HCV) is an important cause of chronic liver disease and is complicated by hepatocellular carcinoma (HCC). Mechanisms whereby the virus promotes cellular transformation are poorly understood. We hypothesized that the guanosine triphosphatase activity encoded in the HCV NS4B protein's nucleotide binding motif (NBM) might play a role in the transformation process. Here we report that NS4B can transform NIH-3T3 cells, leading to tumor formation in vivo. This transformation was independent of co-transfection with activated Ha-ras. Detailed analyses of NS4B mutants revealed that this transforming activity could be progressively inhibited and completely abrogated by increasing genetic impairment of the NS4B nucleotide binding motif.NS4B has in vitro and in vivo tumorigenic potential, and the NS4B transforming activity is indeed mediated by its NBM. Moreover, our results suggest that pharmacological inhibition of the latter might inhibit not only HCV replication but also the associated HCC.

  View details for DOI 10.1002/hep.22108

  View details for PubMedID 18081150

• TBC1D20 is a Rab1 GTPase-activating protein that mediates hepatitis C virus replication JOURNAL OF BIOLOGICAL CHEMISTRY Sklan, E. H., Serrano, R. L., Einav, S., Pfeffer, S. R., Lambright, D. G., Glenn, J. S. 2007; 282 (50): 36354-36361

  Abstract

  Like other viruses, productive hepatitis C virus (HCV) infection depends on certain critical host factors. We have recently shown that an interaction between HCV nonstructural protein NS5A and a host protein, TBC1D20, is necessary for efficient HCV replication. TBC1D20 contains a TBC (Tre-2, Bub2, and Cdc16) domain present in most known Rab GTPase-activating proteins (GAPs). The latter are master regulators of vesicular membrane transport, as they control the activity of membrane-associated Rab proteins. To better understand the role of the NS5A-TBC1D20 interaction in the HCV life cycle, we used a biochemical screen to identify the TBC1D20 Rab substrate. TBC1D20 was found to be the first known GAP for Rab1, which is implicated in the regulation of anterograde traffic between the endoplasmic reticulum and the Golgi complex. Mutation of amino acids implicated in Rab GTPase activation by other TBC domain-containing GAPs abrogated the ability of TBC1D20 to activate Rab1 GTPase. Overexpression of TBC1D20 blocked the transport of exogenous vesicular stomatitis virus G protein from the endoplasmic reticulum, validating the involvement of TBC1D20 in this pathway. Rab1 depletion significantly decreased HCV RNA levels, suggesting a role for Rab1 in HCV replication. These results highlight a novel mechanism by which viruses can hijack host cell machinery and suggest an attractive model whereby the NS5A-TBC1D20 interaction may promote viral membrane-associated RNA replication.

  View details for DOI 10.1074/jbc.M705221200

  View details for PubMedID 17901050

• Mechanisms of resistance to antiviral agents. In Manual of clinical microbiology, 9th edition, Murray PR ed, Baron EJ ed, Jorgensen JH ed, Pfaller MA ed, Tenover FC ed, and Yolken RH ed. American Society of Microbiology. Shafer RW, Einav S, Chou S 2007: 1689-04
• A nucleotide binding motif in hepatitis C virus (HCV) NS4B mediates HCV RNA replication JOURNAL OF VIROLOGY Einav, S., Elazar, M., Danieli, T., Glenn, J. S. 2004; 78 (20): 11288-11295

  Abstract

  Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies.

  View details for PubMedID 15452248

• Prenylation inhibitors: a novel class of antiviral agents JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY Einav, S., Glenn, J. S. 2003; 52 (6): 883-886

  Abstract

  Prenylation is a site-specific lipid modification of proteins. Although first described for a variety of cellular proteins, it has become apparent that viruses can also make use of this post-translational modification provided by their host cells. Depriving a virus access to prenylation can have dramatic effects on the targeted virus's life cycle. Selective pharmacological inhibitors of prenylating enzymes have been developed and shown to have potent antiviral effects in both in vitro and in vivo systems. Because prenylation inhibitors target a host cell function, are available in oral form and are surprisingly well tolerated in human trials, these compounds represent an attractive new class of antiviral agents with potential for broad-spectrum activity. After a brief outline of host cell prenylation pathways, we review below the development of prenylation inhibition as an antiviral strategy applied to a prototype target, hepatitis delta virus (HDV), and discuss the potential application of prenylation inhibitors to a broad range of other viruses.

  View details for DOI 10.1093/jac/dkg490

  View details for PubMedID 14613953

• Immunopathogenesis of hepatitis C virus in the immunosuppressed host. Transplant infectious disease Einav, S., Koziel, M. J. 2002; 4 (2): 85-92

  Abstract

  The prevalence of chronic hepatitis C virus (HCV) infection among various groups of immunosuppressed patients is high. These groups include patients co-infected with human immunodeficiency virus (HIV), recipients of organ transplants, and those with hypogammaglobulinemia. The liver disease in the immunosuppressed host is typically severe with an unusually rapid progression to cirrhosis. This is somewhat paradoxical, as the classical model for HCV-induced liver disease assumes that cell-mediated immune responses induce liver injury. It is likely that a combination of viral-related factors and host-related factors plays a role in this accelerated natural history of HCV. Data are accumulating in immunocompromised hosts that address the immunopathogenesis of liver injury, although there are still fundamental gaps in our understanding of this process. In this review, we will focus on our current understanding of the mechanisms of liver injury and how it relates to the accelerated liver disease progression in immunocompromised hosts.

  View details for PubMedID 12220245

• Complement C4 is protective for lupus disease independent of C3 JOURNAL OF IMMUNOLOGY Einav, S., Pozdnyakova, O. O., Ma, M. H., Carroll, M. C. 2002; 168 (3): 1036-1041

  Abstract

  The role of complement C3 in mediating systemic lupus erythematosus (SLE) was examined using a double-knockout C3(null)C4(null) Fas (CD95)-deficient mouse model. Results from this study reveal significant lymphadenopathy, splenomegaly, elevated titers of anti-nuclear Abs and anti-dsDNA Abs, an increased number of anti-dsDNA-producing cells in ELISPOT assay, as well as severe glomerulonephritis in the double-deficient mice. Based on these clinical, serological, and histological parameters, we find that autoimmune disease in the double-knockout group is similar in severity to that in C4(null) lpr mice, but not to that in C3(null) lpr mice. The development of severe SLE in the absence of both classical and alternative complement pathways suggests that it is the absence of C4, and not the presence of C3, that is critical in SLE pathogenesis. Thus, complement C4 provides an important protective role against the development of SLE.

  View details for Web of Science ID 000173429300010

  View details for PubMedID 11801636