title: GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.
abstract: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.IMPORTANCERecent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.
authors.affiliation: Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.|Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, India.
authors.forename: Minu
authors.initials: M
authors.lastname: Nain
authors.affiliation: National Brain Research Centre, Manesar, Haryana, India.
authors.forename: Sriparna
authors.initials: S
authors.lastname: Mukherjee
authors.affiliation: Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
authors.forename: Sonali Porey
authors.initials: SP
authors.lastname: Karmakar
authors.affiliation: Research Centre for Infectious Diseases, Department of Molecular and Cellular Biology, University of Adelaide, Adelaide, Australia.
authors.forename: Adrienne W
authors.initials: AW
authors.lastname: Paton
authors.affiliation: Research Centre for Infectious Diseases, Department of Molecular and Cellular Biology, University of Adelaide, Adelaide, Australia.
authors.forename: James C
authors.initials: JC
authors.lastname: Paton
authors.affiliation: Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi, India.
authors.forename: M Z
authors.initials: MZ
authors.lastname: Abdin
authors.affiliation: National Brain Research Centre, Manesar, Haryana, India.
authors.forename: Anirban
authors.initials: A
authors.lastname: Basu
authors.affiliation: Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India manjula@thsti.res.in vrati@thsti.res.in.
authors.forename: Manjula
authors.initials: M
authors.lastname: Kalia
authors.affiliation: Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India manjula@thsti.res.in vrati@thsti.res.in.|Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India.
authors.forename: Sudhanshu
authors.initials: S
authors.lastname: Vrati
mesh_terms: D000818:Animals; D002460:Cell Line; D004664:Encephalitis Virus, Japanese / Q000502:physiology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000821:virology; D011485:Protein Binding; D014759:Viral Envelope Proteins / Q000378:metabolism; D053586:Virus Internalization; D014779:Virus Replication
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D014759:Viral Envelope Proteins
keywords: Japanese encephalitis virus; flavivirus; host-cell interactions; receptors; viral replication; virus entry