Tick‐Borne Flaviviruses Activate the IRE1 Stress Pathway in a Virus‐ and Cell‐Type Dependent Manner Promoting Virus Replication

Abstract

Tick-borne flaviviruses (TBFV) are transmitted by either tick-bite or consumption of raw milk products from infected ruminants. After crossing the blood-brain barrier, the virus enters the central nervous system (CNS) leading to neural inflammation and causing neurological complications. Individual viruses within this group, show differences in tissue tropism and pathogenicity. The mechanisms behind these differences are poorly understood, but likely result from a mix of direct viral replication and cellular responses to infection. Flavivirus replication is ER-associated and uses the cellular machinery for viral protein synthesis. The increased protein burden results in the activation of the unfolded protein response (UPR). The IRE1 pathway is hypothesized to support flavivirus replication by increasing protein and lipid biogenesis. We investigated the ER stress response following flavivirus infection in relevant target cell types as well as the effect of ER stress inhibition on virus replication. For this purpose, human astrocyte, neuronal and intestinal cell lines were infected with tick-borne encephalitis (TBEV) strains Neudörfl and HB171 as well as Langat virus (LGTV) and the rate of virus replication and activated UPR pathways in these cells were investigated. All three viruses activated the ER stress response in a virus- and cell-type dependent manner with IRE1 being the prominent pathway. The upregulation of X-box binding protein 1 (XBP1) as marker protein for the IRE1 pathway, differed between the cell lines and showed varying levels of intensities. The levels of activation of the IRE1 pathway among the three viruses correlated with their described levels of pathogenicity. As we found IRE1 to be the most strongly induced pathway, we further investigated the effect of three different IRE1 inhibitors on virus replication and cell viability during TBFV infection. Our data showed that TBFV replication was significantly reduced upon inhibition of the kinase and RNAse activity of IRE1, either combined or alone. These results confirm the supporting role of the IRE1 pathway for TBFV infection in relevant viral target cells and suggest that virus and cell-type dependent differences in UPR induction may affect the outcome of the infection.