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.

Results obtained from the subcellular fraction of HCV-infected cells show that the majority of membrane-bound viral RNA is associated with the microsomal fraction, and the concentration of NS5B in this fraction suggests this RNA fraction includes replication complexes. These data are consistent with previous studies reporting that positive-strand RNA virus replication complexes are formed in association with ER-like membranes (Reviewed in [57] ). The microsomal fractions are also predicted to contain positive-strand RNA being translated into HCV polyprotein on membrane-bound ribosomes [6, [11] [12] [13] . However, the processes of viral RNA replication and translation have been suggested to occur in distinct locations [48, 57, 58] . To further assess the spatial relationship between translation and the MW, we examined the distribution of ribosomes in HCV-infected cells using immunofluorescence microscopy. We stained uninfected or HCV-infected Huh7.5 cells with antibodies directed against the S6 protein component of the 40S ribosomal subunit. In uninfected cells, ribosomes were distributed throughout the cytoplasm ( Fig 5A) . However, in infected cells the vast majority of the S6 protein was excluded from regions of the cytoplasm containing the bulk of the HCV core protein (Fig 5A) . The separation of the two fluorescent signals was also supported by negative Pearson's correlation coefficients (Fig 5) . The same general phenotype was observed when we compared the localization of the S6 protein to that of NS5A or positive-strand HCV RNA (Fig 5B and 5C) , consistent with the conclusion that the bulk of the ribosomes are located in regions of the cell separate from HCV replication and assembly complexes. Consistent with our observations in Fig 3B showing that a portion of the viral positive-strand RNA colocalizes with RIG-I, Manders Overlap Coefficient's revealed that, while the majority of the positive-strand RNA (76% of the fluorescent signal) was found in regions lacking ribosomal signal, a smaller pool of positivestrand RNA (24% of the fluorescent signal) overlapped with the ribosomal S6 signal (Fig 5C) .