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.

We also examined the effect of expressing various RIG-I constructs, including RIG-I, NLS-RIG-I, NLS-RIG-I-K270A, or SLN-RIG-I (SLN encodes a reverse sequence, non-functional NLS) on virus production. Similar levels of each of these constructs were detected in transfected cell populations (S5A Fig). Furthermore, the expression of each RIG-I construct alone stimulated only minimal, statistically insignificant, changes in the levels of immune stimulated genes compared to those expressing wild type RIG-I, indicating that the NLS tag has little or no effect on RIG-I-mediated immune activation (S5B Fig). We then tested the consequences of expressing these various RIG-I constructs in HCV-infected cells. For this analysis, cells were infected with HCV followed by transfection with each of the four RIG-I constructs, and total viral RNA levels were determined by real-time PCR. As predicted, increased cellular levels of RIG-I caused a small, but significant decrease in HCV RNA levels (Fig 8C) .