Valosin-Containing Protein/p97 Plays Critical Roles in the Japanese Encephalitis Virus Life Cycle

Abstract

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. Importance JEV 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.

How Viruses Use the VCP/p97 ATPase Molecular Machine

“…Recent [53]. Additional characterization indicated that the VCP inhibitor prevented JEV from leaving clathrin-coated vesicles and interfered with capsid protein degradation [53], in agreement with the idea that ATPase activity is required for uncoating of flaviviruses.…”

“…Furthermore, the role of VCP/p97 is evolutionarily conserved since inhibitors also blocked pre-replication events of YFV in mosquito cells [40]. Recent [53]. Additional characterization indicated that the VCP inhibitor prevented JEV from leaving clathrin-coated vesicles and interfered with capsid protein degradation [53], in agreement with the idea that ATPase activity is required for uncoating of flaviviruses.…”

“…Members of the Flaviviridae family, including JEV, WNV, ZIKV, DENV, and hepatitis C virus (HCV), also depend on p97 for their replication. Depletion of the VCP adapter, UFD1, did not affect JEV entry, but reduced viral RNA levels [53]. Transfection of JEV RNA into HeLa cells was used to bypass the entry step and revealed that viral replication was restricted in the presence of VCP-specific siRNA.…”

“…Transfection of JEV RNA into HeLa cells was used to bypass the entry step and revealed that viral replication was restricted in the presence of VCP-specific siRNA. Furthermore, p97 co-localized near the nucleus with nonstructural proteins NS1 and NS5, but not capsid [53]. All of the flavivirus nonstructural proteins are involved in RNA replication [77].…”

“…Immunoprecipitation of tagged forms of NS3 and NS5 with VCP only occurred in infected cells, suggesting that other viral factors were required for nonstructural protein interactions with p97. WNV genomic replication also was decreased in the absence of p97 [53]. Using p97-specific siRNAs in combination with a DNA-replicon system to bypass viral entry, knockdown decreased WNV RNA levels [39].…”