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.

25 μM ivermectin or 40 μM importazole for 4 hours and then examined. A) HCV NS5A was detected using specific antibodies and indirect immunofluorescence and the GFP fusions were visualized directly using fluorescence confocal microscopy. DNA was stained with DAPI (blue). Boxed regions in the middle row of both panels outline the area of magnification presented in the bottom rows. Scale bars represent 5 μm. Pearson's correlation coefficients shown in the merge images of HCV-infected cells were calculated using Coloc2 software in ImageJ and represent overlap between the red and green fluorescent channels in the indicated image. B) Pearson's correlation coefficients were determined to assess overlap of HCV NS5SA and GFP signals in confocal images derived from HCV-infected cells expressing the indicated RIG construct. The values presented represent an average over 10 images (at least 15 cells) and the error bars indicate standard error. Significance was evaluated using t-tests and p-values less than 0.05 (*) are indicated. Strikingly, introduction of the NLS-RIG-I construct lead to significant further decrease in levels of HCV RNA as compared to wild-type RIG-I. By contrast, no changes were observed in cells transfected with the SLN-RIG-I or NLS-RIG-I-K270A expression constructs. These results further implicate the nuclear transport machinery as a regulator of access to sites of virus replication and assembly.