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Processing of capsid protein by cathepsin L plays a crucial role in replication of Japanese encephalitis virus in neural and macrophage cells.

ABSTRACT: The flavivirus capsid protein not only is a component of nucleocapsids but also plays a role in viral replication. In this study, we found a small capsid protein in cells infected with Japanese encephalitis virus (JEV) but not in the viral particles. The small capsid protein was shown to be generated by processing with host cysteine protease cathepsin L. An in vitro cleavage assay revealed that cathepsin L cleaves the capsid protein between amino acid residues Lys(18) and Arg(19), which are well conserved among the mosquito-borne flaviviruses. A mutant JEV resistant to the cleavage of the capsid protein by cathepsin L was generated from an infectious cDNA clone of JEV by introducing a substitution in the cleavage site. The mutant JEV exhibited growth kinetics similar to those of the wild-type JEV in monkey (Vero), mosquito (C6/36), and porcine (PK15) cell lines, whereas replication of the mutant JEV in mouse macrophage (RAW264.7) and neuroblastoma (N18) cells was impaired. Furthermore, the neurovirulence and neuroinvasiveness of the mutant JEV to mice were lower than those of the wild-type JEV. These results suggest that the processing of the JEV capsid protein by cathepsin L plays a crucial role in the replication of JEV in neural and macrophage cells, which leads to the pathogenesis of JEV infection.

SUBMITTER: Mori Y

PROVIDER: S-EPMC1951360 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Processing of capsid protein by cathepsin L plays a crucial role in replication of Japanese encephalitis virus in neural and macrophage cells.

Mori Yoshio Y Yamashita Tetsuo T Tanaka Yoshinori Y Tsuda Yoshimi Y Abe Takayuki T Moriishi Kohji K Matsuura Yoshiharu Y

Journal of virology 20070606 16

The flavivirus capsid protein not only is a component of nucleocapsids but also plays a role in viral replication. In this study, we found a small capsid protein in cells infected with Japanese encephalitis virus (JEV) but not in the viral particles. The small capsid protein was shown to be generated by processing with host cysteine protease cathepsin L. An in vitro cleavage assay revealed that cathepsin L cleaves the capsid protein between amino acid residues Lys(18) and Arg(19), which are well ...[more]

PMID: 17553875

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Project description:The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Project description: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.

Project description:BackgroundThe low immunogenicity of neural stem/progenitor cells (NSPCs) coupled with negligible expression of MHC antigens has popularized their use in transplantation medicine. However, in an inflammatory environment, the NSPCs express costimulatory molecules and MHC antigens, and also exhibit certain immunomodulatory functions. Since NSPCs are the cellular targets in a number of virus infections both during postnatal and adult stages, we wanted to investigate the immunological properties of these stem cells in response to viral pathogen.Methodology/principal findingsWe utilized both in vivo mouse model and in vitro neurosphere model of Japanese encephalitis virus (JEV) infection for the study. The NSPCs residing in the subventricular zone of the infected brains showed prominent expression of MHC-I and costimulatory molecules CD40, CD80, and CD86. Using Flow cytometry and fluorescence microscopy, we observed increased surface expression of co-stimulatory molecule and MHC class I antigen in NSPCs upon progressive JEV infection in vitro. Moreover, significant production of pro-inflammatory cyto/chemokines was detected in JEV infected NSPCs by Cytokine Bead Array analysis. Interestingly, NSPCs were capable of providing functional costimulation to allogenic T cells and JEV infection resulted in increased proliferation of allogenic T cells, as detected by Mixed Lymphocyte reaction and CFSE experiments. We also report IL-2 production by NSPCs upon JEV infection, which possibly provides mitogenic signals to T cells and trigger their proliferation.Conclusion/significanceThe in vivo and in vitro findings clearly indicate the development of immunogenicity in NSPCs following progressive JEV infection, in our case, JEV infection. Following a neurotropic virus infection, NSPCs possibly behave as immunogenic cells and contribute to both the innate and adaptive immune axes. The newly discovered immunological properties of NSPCs may have implications in assigning a new role of these cells as non-professional antigen presenting cells in the central nervous system.

Dataset Information

Processing of capsid protein by cathepsin L plays a crucial role in replication of Japanese encephalitis virus in neural and macrophage cells.

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Processing of capsid protein by cathepsin L plays a crucial role in replication of Japanese encephalitis virus in neural and macrophage cells.

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