Valosin-containing protein/p97 plays critical roles in the Japanese encephalitis virus life cycle.

Host factors provide critical support for every aspect of the virus life cycle. We recently identified the valosin-containing protein (VCP)/p97, an abundant cellular ATPase with diverse cellular functions, as a host factor important for Japanese encephalitis virus (JEV) replication. In cultured cells, using siRNA-mediated protein depletion and pharmacological inhibitors, we show that VCP is crucial for replication of three flaviviruses: JEV, Dengue, and West Nile viruses. An FDA-approved VCP inhibitor, CB-5083, extended survival of mice in the animal model of JEV infection. While VCP depletion did not inhibit JEV attachment on cells, it delayed capsid degradation, potentially through the entrapment of the endocytosed virus in clathrin-coated vesicles (CCVs). Early during infection, VCP-depleted cells showed an increased colocalization of JEV capsid with clathrin, and also higher viral RNA levels in purified CCVs. We show that VCP interacts with the JEV nonstructural protein NS5 and is an essential component of the virus replication complex. The depletion of the major VCP cofactor UFD-1 also significantly inhibited JEV replication. Mechanistically, thus, VCP affected two crucial steps of the JEV life cycle - nucleocapsid release and RNA replication. Our study establishes VCP as a common host factor with a broad antiviral potential against flaviviruses.ImportanceJEV is the leading cause of viral encephalitis epidemics in South-east Asia, affecting majorly children with high morbidity and mortality. Identification of host factors is thus essential for the rational design of anti-virals that are urgently need as therapeutics. Here we have identified the VCP protein as one such host-factor. This protein is highly abundant in cells and engages in diverse functions and cellular pathways by its ability to interact with different co-factors. Using siRNA mediated protein knockdown, we show that this protein is essential for release of the viral RNA into the cell so that it can initiate replication. The protein plays a second crucial role for the formation of the JEV replication complex. FDA-approved drugs targeting VCP show enhanced mouse survival in JE model of disease, suggesting that this could be a druggable target for flavivirus infections.

Proteins involved in actin filament organization are key host factors for Japanese encephalitis virus life-cycle in human neuronal cells.

Multiple membrane trafficking networks operate in the eukaryotic cell and are hijacked by viruses to establish infection. Recent studied have highlighted that viruses can exploit distinct pathways depending on the cell type. Japanese encephalitis virus (JEV), a neurotropic flavivirus, can infect neuronal cells through a clathrin-independent endocytic mechanism. To further characterize the membrane trafficking requirements for JEV infection of neuronal cells, we have performed a RNA interference-based study targeting 136 proteins in the human cell line IMR-32. Through quantitative RT-PCR and plaque assays we have validated that JEV infection in neuronal cells was independent of clathrin, and identified host-factors that were crucial for establishment of infection. Several of these proteins were involved in regulation of actin filament organization such as RHOA, RAC1, proteins of the ARP2/3 complex and N-WASP family, LIMK1, PAK1 and ROCK2. The small molecule inhibitors of ARP2/3 complex, CK-548 and of the N-WASP, Wiskostatin inhibited virus replication highlighting the important roles of these proteins in the virus life-cycle. We also identified ATG12, BECN1, VAPA, VAPB and VCP proteins as crucial host-factors for JEV replication across epithelial and neuronal cell lineages.

Membrane trafficking RNA interference screen identifies a crucial role of the clathrin endocytic pathway and ARP2/3 complex for Japanese encephalitis virus infection in HeLa cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is one of the leading global causes of virus-induced encephalitis. The infectious life-cycle of viruses is heavily dependent on the host membrane trafficking network. Here, we have performed a RNA-interference-based screen using a siRNA panel targeting 136 membrane trafficking proteins to identify the key regulators of JEV infection in HeLa cells. We identified 35 proteins whose siRNA depletion restricts JEV replication by over twofold. We observe that JEV infection in HeLa cells is largely dependent on components of the clathrin-mediated endocytic (CME) pathway. Proteins involved in actin-filament-based processes, specifically CDC42 and members of the ARP2/3 complex are crucial for establishment of infection. Pharmacological pertubations of actin polymerization, a small molecule inhibitor of actin nucleation by the ARP2/3 complex - CK-548 - and the inhibitor of neural Wiskott-Aldrich syndrome proteins- Wiskostatin- inhibited JEV replication, highlighting the important role of the dynamic actin network. Other proteins involved in cargo-recognition for CME and endomembrane system organization were also validated as essential host factors for virus replication.

A screen for novel hepatitis C virus RdRp inhibitor identifies a broad-spectrum antiviral compound.

Hepatitis C virus (HCV) is a global pathogen and infects more than 185 million individuals worldwide. Although recent development of direct acting antivirals (DAA) has shown promise in HCV therapy, there is an urgent need for the development of more affordable treatment options. We initiated this study to identify novel inhibitors of HCV through screening of compounds from the National Cancer Institute (NCI) diversity dataset. Using cell-based assays, we identified NSC-320218 as a potent inhibitor against HCV with an EC50 of 2.5 µM and CC50 of 75 µM. The compound inhibited RNA dependent RNA polymerase (RdRp) activity of all six major HCV genotypes indicating a pan-genotypic effect. Limited structure-function analysis suggested that the entire molecule is necessary for the observed antiviral activity. However, the compound failed to inhibit HCV NS5B activity in vitro, suggesting that it may not be directly acting on the NS5B protein but could be interacting with a host protein. Importantly, the antiviral compound also inhibited dengue virus and hepatitis E virus replication in hepatocytes. Thus, our study has identified a broad-spectrum antiviral therapeutic agent against multiple viral infections.

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes.

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.

Japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism.

Japanese encephalitis virus (JEV) is a mosquito-borne pathogenic flavivirus responsible for acute viral encephalitis in humans. The cellular entry of JEV is poorly characterized in terms of molecular requirements and pathways. Here we present a systematic study of the internalization mechanism of JEV in fibroblasts and neuroblastoma cells. To verify the roles of distinct pathways of cell entry, we used fluorescently labeled virus particles, a combination of pharmacological inhibitors, RNA interference (RNAi), and dominant-negative (DN) mutants of regulatory proteins involved in endocytosis. Our study demonstrates that JEV infects fibroblasts in a clathrin-dependent manner, but it deploys a clathrin-independent mechanism to infect neuronal cells. The clathrin-independent pathway requires dynamin and plasma membrane cholesterol. Virus binding to neuronal cells leads to rapid actin rearrangements and an intact and dynamic actin cytoskeleton, and the small GTPase RhoA plays an important role in viral entry. Immunofluorescence analysis of viral colocalization with endocytic markers showed that JEV traffics through Rab5-positive early endosomes and that release of the viral nucleocapsid occurs at the level of the early and not the late endosomes.