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.IMPORTANCE Recent 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.

Japanese encephalitis virus (JEV), which is a causative agent of sporadic encephalitis, harbours itself inside the neural stem/progenitor cells. It is a well-known fact that JEV infects neural stem/progenitor cells and decreases their proliferation capacity. With mass spectrometry-based quantitative proteomic study, it is possible to reveal the impact of virus on the stem cells at protein level. Our aim was to perceive the stem cell proteomic response upon viral challenge. We performed a two-dimensional gel electrophoresis-based proteomic study of the human neural stem cells (hNS1 cell line) post JEV infection and found that 13 proteins were differentially expressed. The altered proteome profile of hNS1 cell line revealed sustained endoplasmic reticulum stress, which deteriorated normal cellular activities leading to cell apoptosis. The proteomic changes found in hNS1 cell line were validated in vivo in the subventricular zone of JE infected BALB/c mice. Congruent alterations were also witnessed in multipotent neural precursor cells isolated from human foetus and in autopsy samples of human brain clinically diagnosed as cases of JE patients. Endoplasmic reticulum resident chaperone GRP78, mitochondrial protein Prohibitin and heterogeneous nuclear ribonucleoprotein hnRNPC (C1/C2) have been shown to interact with viral RNA. Hence it is proposed that these are the principle candidates governing endoplasmic reticulum stress-induced apoptosis in JEV infection.

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MicroRNAs (miRNAs) released from the activated microglia upon neurotropic virus infection may exacerbate the neuronal damage. Here, we identified let‐7a and let‐7b (let‐7a/b) as one of the essential miRNAs over‐expressed upon Japanese Encephalitis virus (JEV) infection and released in the culture supernatant of the JEV‐infected microglial cells through extracellular vesicles. The let‐7a/b was previously reported to modulate inflammation in microglial cells through Toll‐like receptor 7 (TLR7) pathways; although their role in accelerating JEV pathogenesis remain unexplored. Therefore, we studied the role of let‐7a/b in modulating microglia‐mediated inflammation during JEV infection and investigated the effect of let‐7a/b‐containing exosomes on primary neurons. To this end, we examined let‐7a/b and NOTCH signaling pathway in TLR7 knockdown (KD) mice. We observed that TLR7 KD or inhibition of let‐7a/b suppressed the JEV‐induced NOTCH activation possibly via NF‐κB dependent manner and subsequently, attenuated JEV‐induced TNFα production in microglial cells. Furthermore, exosomes secreted from let‐7a/b over‐expressed microglia when transferred to uninfected mice brain induced caspase activation. Exosomes secreted from virus‐infected or let‐7a/b over‐expressed microglia when co‐incubated with mouse neuronal (Neuro2a) cells or primary cortical neurons also facilitated caspase activation leading to neuronal death. Thus, our results provide evidence for the multifaceted role of let‐7a/b miRNAs in JEV pathogenesis. Let‐7a/b can interact with TLR7 and NOTCH signaling pathway and enhance TNFα release from microglia. On the other hand, the exosomes secreted by JEV‐infected microglia can activate caspases in uninfected neuronal cells which possibly contribute to bystander neuronal death. Cover Image for this issue: doi: .