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 postulate that exclusion of RLRs from the MW contributes to masking the viral genome from the cell's innate immune response. Since our data show that adding an NLS to the RLRs allows the fusion proteins to use the nuclear transport machinery and access the MW, we examined the effects of NLS-RLR entry into the MW on innate immune activation in HCVinfected cells. To assess immune response, we examined the localization of IRF-3 in uninfected or HCV-infected cells expressing either RIG-I or NLS-RIG-I (Fig 8A and 8B) . The re-localization of IRF-3 to the nucleus is an initial step in RLR-mediated immune activation [63] . Therefore, we used immunofluorescence microscopy to monitor nuclear translocation of IRF-3 to determine immune activation in cells expressing the RIG-I constructs. This approach allowed us to assess activation of the immune response in individual cells determined to be both HCVinfected or uninfected and transfected with the RIG-I fusion constructs. In uninfected cells, we found that the expression of RIG-I or NLS-RIG-I did not significantly alter the localization of IRF-3 compared to that of untransfected cells, with IRF-3 appearing largely cytoplasmic and excluded from the nucleus (Fig 8A and 8B ). However, in HCV-infected cells, the nuclear localization of IRF-3 was significantly increased in cells expressing NLS-RIG-I as compared to those expressing RIG-I or untransfected cells (Fig 8A and 8B) . We conclude from these results that the expression of NLS-RIG-I increases immune activation in HCV-infected cells to a greater extent than overexpression of wild-type RIG-I.