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.

These data are consistent with previous studies documenting a role for the nuclear transport machinery in controlling access of molecules to HCV-induced MW [37] . We evaluated this further by examining whether the ability of the NLS-RIG-I-K270A construct to access regions occupied by the MW required nuclear transport factors. For this analysis, we examined the effects of two small molecule inhibitors, ivermectin and importazole [61, 62] , that specifically block the transport function of the SV40 large T antigen NLS cognate nuclear import receptor, the importin α/β dimer. HCV-infected cells expressing GFP-tagged RIG-I-K270A or GFPtagged NLS-RIG-I-K270A were treated with either ivermectin or importazole for 3 hours and then evaluated by confocal microscopy. In the absence of the inhibitors, the degree of co-localization between RIG-I-K270A or NLS-RIG-I-K270A and NS5A in HCV-infected cells was similar to that presented Figs 1 and 6 (Fig 7 top panels) , with the NLS-RIG-I-K270A construct and NS5A showing a positive Pearson correlation coefficient. However, when these cells were treated with ivermectin or importazole, the overlap between NLS-tagged RIG-I-I-K270A and NS5A was significantly decreased and a negative Pearson correlation coefficient was observed. By contrast, neither drug affected the distribution or the degree of overlap between RIG-I-K270A and NS5A (Fig 7) . These results further support a model in which the nuclear transport machinery regulates access of proteins to regions within the MW.