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.

Huh7.5 cells were transfected with constructs encoding either GFP-tagged RIG-I or GFP-tagged NLS-RIG-I 2 days after HCV infection. On day 4 after infection, the localization of IRF-3 (grey) was examined using specific antibodies and indirect immunofluorescence and the GFP fusions (green) were visualized directly using fluorescence confocal microscopy. In addition, to identify HCV-infected cells amongst those expressing the RIG-I constructs, cells were stained with antibodies directed against the HCV core protein (red). DNA was stained with DAPI (blue) and scale bars represent 5 μm. B) The ratio of nuclear to cytoplasmic fluorescence signal arising from IRF-3 indirect immunofluorescence was calculated using ImageJ for > 30 uninfected or HCV-infected cells alone or expressing either GFP-tagged RIG-I or GFP-tagged NLS-RIG-I. Significance was evaluated using t-tests and p-values less than 0.05 (*), 0.01 (**) or 0.001 (*) are indicated. C) Uninfected and HCV-infected Huh7.5 cells were transfected with constructs encoding for GFP-RIG-I, GFP-NLS-RIG-I, GFP-SLN-RIG-I, or GFP-NLS-RIG-I-K270A 1 day after HCV infection. 3 days after infection, cells were harvested and levels of intracellular HCV RNA were determined by qPCR. Fold change of intracellular HCV RNA was calculated relative to HCV-infected cells transfected with constructs encoding for GFP alone.