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

2021 ◽  
Author(s):  
Sapna Sehrawat ◽  
Renu Khasa ◽  
Arundhati Deb ◽  
Surendra Kumar Prajapat ◽  
Suvadip Mallick ◽  
...  
Keyword(s):  
Life Cycle ◽  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Host Factor ◽  
Encephalitis Virus ◽  
Host Factors ◽  
Viral Rna ◽  
High Morbidity ◽  
Replication Complex ◽  
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. 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.

scholarly journals Screening of Natural Extracts for Inhibitors against Japanese Encephalitis Virus Infection

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Jiao Guo ◽  
Xiaoying Jia ◽  
Yang Liu ◽  
Shaobo Wang ◽  
Junyuan Cao ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Antiviral Agents ◽  
Drug Repurposing ◽  
Encephalitis Virus ◽  
Morbidity And Mortality ◽  
High Morbidity ◽  
Japanese Encephalitis Virus Infection ◽  
Natural Extracts ◽  
Effective Drugs

ABSTRACT The mosquito-borne Japanese encephalitis virus (JEV) causes serious illness worldwide that is associated with high morbidity and mortality. Currently, there are no effective drugs approved for the treatment of JEV infection. Drug-repurposing screening is an alternative approach to discover potential antiviral agents. In this study, high-content screening (HCS) of a natural extracts library was performed, and two hit FDA-approved Na+/K+-ATPase inhibitors, ouabain and digoxin, were identified as having robust efficiency against JEV infection with the selectivity indexes over 1,000. The results indicated that ouabain and digoxin blocked the JEV infection at the replication stage by targeting the Na+/K+-ATPase. Furthermore, it was proven that ouabain significantly reduced the morbidity and mortality caused by JEV in a BALB/c mouse model. This work demonstrated that Na+/K+-ATPase could serve as the target of treatment of JEV infection, and ouabain has the potential to be developed as an effective anti-JEV drug.

scholarly journals CRISPR screening of porcine sgRNA library identifies host factors associated with Japanese encephalitis virus replication

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Changzhi Zhao ◽  
Hailong Liu ◽  
Tianhe Xiao ◽  
Zichang Wang ◽  
Xiongwei Nie ◽  
...  
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Species ◽  
Encephalitis Virus ◽  
Host Factors ◽  
Protein Coding ◽  
Guide Rnas ◽  
Screening Platform ◽  
The Many ◽  
Genome Scale

Abstract Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.

scholarly journals Cell Type-Dependent RNA Recombination Frequency in the Japanese Encephalitis Virus

2014 ◽  
Vol 2014 ◽  
pp. 1-9
Author(s):  
Wei-Wei Chiang ◽  
Ching-Kai Chuang ◽  
Mei Chao ◽  
Wei-June Chen
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Mammalian Cells ◽  
Viral Evolution ◽  
Cell Types ◽  
Encephalitis Virus ◽  
Viral Rna ◽  
Rna Recombination ◽  
Mosquito Cells ◽  
Rna Fragments

Japanese encephalitis virus (JEV) is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously.

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