The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

A caveat of the experiments examining the exogenous expression of RLRs is that overexpression of the active form of RIG-I in HCV-infected Huh7.5 cells leads to immune activation, which impedes viral replication and could alter the structure of the MW [29] . To avoid this complication, we used a point mutant of RIG-I (RIG-I-K270A) that can still bind to viral RNA but lacks helicase activity necessary for tracking along the RNA strand and downstream immune signalling [44] . We observed that the subcellular localization of Rig-I-K270A was similar to that of the RIG-I in uninfected and HCV-infected cells (Fig 1C and S1B Fig) . Furthermore, RIG-I-K270A was also reduced in regions of the cytoplasm where HCV NS4B and core were concentrated (Fig 1C) . Rig-I-K270A signal was also absent from cytoplasmic regions containing lipid droplets, which are induced by HCV infection and generally associated with core protein, as well as from regions surrounding lipid droplets (S1C Fig). HAV infection induces cytoplasmic compartments lacking RIG-I All positive-strand RNA viruses reorganize host cell membranes, often forming distinct replication complexes [9, 45] . To test if the exclusion of RLRs from regions occupied by viral replication compartments is a phenomenon displayed by other positive-strand RNA viruses, we examined the localization of RIG-I-K270A in hepatitis A virus (HAV)-infected cells. As shown in Fig 2, in HAV-infected Huh7.5 cells, we observed exclusion of RIG-I-K270A from cytoplasmic regions containing HAV capsid protein or viral dsRNA, results similar to that seen in HCV-infected cells. These data are consistent with the proposed idea that virus-induced cytoplasmic compartments may represent a common immune evasion strategy of positivestrand RNA viruses.