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Stringent regulation of antiviral signaling and cellular autophagy is critical for the host response to virus infection. However, little is known how these cellular processes are regulated in the absence of type I interferon signaling. Here, we show that ATF3 is induced following Japanese encephalitis virus (JEV) infection, and regulates cellular antiviral and autophagy pathways in the absence of type I interferons in mouse neuronal cells. We have identified new targets of ATF3 and show that it binds to the promoter regions of Stat1, Irf9, Isg15 and Atg5 thereby inhibiting cellular antiviral signaling and autophagy. Consistent with these observations, ATF3-depleted cells showed enhanced antiviral responses and induction of robust autophagy. Furthermore, we show that JEV replication was significantly reduced in ATF3-depleted cells. Our findings identify ATF3 as a negative regulator of antiviral signaling and cellular autophagy in mammalian cells, and demonstrate its important role in JEV life cycle.

Endoplasmic reticulum (ER) stress and autophagy are key cellular responses to RNA virus infection. Recent studies have shown that Japanese encephalitis virus (JEV)-induced autophagy negatively influences virus replication in mouse neuronal cells and embryonic fibroblasts, and delays virus-induced cell death. Here, we evaluated the role of ER stress pathways in inducing autophagy during JEV infection. We observed that JEV infection of neuronal cells led to activation of all three sensors of ER stress mediated by eIF2α/PERK, IRE1/XBP1 and ATF6. The kinetics of autophagy induction as monitored by levels of SQSTM1 and LC3-II paralleled activation of ER stress. Inhibition of the eIF2α/PERK pathway by siRNA-mediated depletion of proteins and by the PERK inhibitor had no effect on autophagy and JEV replication. However, depletion of XBP1 and ATF6, alone or in combination, prevented autophagy induction and significantly enhanced JEV-induced cell death. JEV-infected cells depleted of XBP1 or ATF6 showed reduced transcription of ER chaperones, ERAD components and autophagy genes, resulting in reduced protein levels of the crucial autophagy effectors ATG3 and BECLIN-1. Conversely, pharmacological induction of ER stress in JEV-infected cells further enhanced autophagy and reduced virus titres. Our study thus demonstrates that a crucial link exists between the ER stress pathways and autophagy in virus-infected cells, and that these processes are highly regulated during virus infection.

Diphenyleneiodonium (DPI) and N-acetyl- l -cysteine (NAC), two widely used anti-oxidants, were employed to evaluate the role of oxidative stress in Japanese encephalitis virus (JEV) induced autophagy, stress responses and replication. DPI and NAC exerted opposite effects on ROS levels in JEV infected mouse neuronal cells (Neuro2a), mouse embryonic fibroblasts (MEFs) and human epithelial cells (HeLa). While NAC effectively quenched ROS, DPI enhanced ROS levels, suggesting that DPI induces oxidative stress in JEV infected cells. DPI treatment of JEV infected Neuro2a cells further blocked autophagy induction and activation of all three arms of the ER stress pathway, and, inhibited virus particle release. Autophagy induction in JEV infection has been previously shown to be linked to the activation of XBP1 and ATF6 ER stress sensors. Our data suggests that DPI mediated block of autophagy is a result of inhibition of ER stress responses and is not associated with an anti-oxidative effect. Since DPI has a wide inhibitory potential for all Flavin dependent enzymes, it is likely that the signalling pathways for ER stress and autophagy during JEV infection are modulated by DPI sensitive enzymes. [ABSTRACT FROM AUTHOR]

Japanese encephalitis virus (JEV) is a flavivirus , spread by the bite of carrier Culex mosquitoes. The subsequent disease caused is Japanese encephalitis (JE), which is the leading global cause of virus-induced encephalitis. The disease is predominant in the entire Asia-Pacific region with the potential of global spread. JEV is highly neuroinvasive with symptoms ranging from mild fever to severe encephalitis and death. One-third of JE infections are fatal, and half of the survivors develop permanent neurological sequelae. Disease prognosis is determined by a series of complex and intertwined signaling events dictated both by the virus and the host. All flaviviruses , including JEV replicate in close association with ER derived membranes by channelizing the protein and lipid components of the ER. This leads to activation of acute stress responses in the infected cell-oxidative stress, ER stress, and autophagy. The host innate immune and inflammatory responses also enter the fray, the components of which are inextricably linked to the cellular stress responses. These are especially crucial in the periphery for dendritic cell maturation and establishment of adaptive immunity. The pathogenesis of JEV is a combination of direct virus induced neuronal cell death and an uncontrolled neuroinflammatory response. Here we provide a comprehensive review of the JEV life cycle and how the cellular stress responses dictate the pathobiology and resulting immune response. We also deliberate on how modulation of these stress pathways could be a potential strategy to develop therapeutic interventions, and define the persisting challenges. [ABSTRACT FROM AUTHOR]