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 observed an enrichment of viral polymerase NS5B in the microsomal membrane fractions, but only low levels of core and NS3. By contrast, core and NS3, but not NS5B, were enriched in the MAM fraction (Fig 4A and 4B ). NS5A was observed at similar levels in both the microsomal and MAM fractions ( Fig 4B) . Thus, key components of viral replication were enriched within microsomal membrane fractions, while assembly center components were found in the MAM fraction. We also evaluated the levels of total viral RNA and infections virus present in the various membrane fractions and cytosol ( Fig 4C) . Viral RNA and infectious virus were detected in the cytosol, presumably arising from membrane fragmentation that occurs during cell lysis. Within the membrane fractions, we observed that higher levels of total viral RNA were present in the microsomal fraction. However, approximately double the percentage of infectious virus was present in the MAM fraction. These fractionation results lead us to conclude that distinct replication and assembly compartments are present in HCVinfected cells, with the microsomal membrane fraction enriched for viral replication complexes Total cell lysates isolated from uninfected (UN) or HCV-infected (HCV) Huh7.5 cells were subjected to subcellular fractionation. A and B) Western blotting with antibodies specific for the indicated proteins was used to evaluate their relative amounts in various subcellular membrane fractions. Indicated above the blot image are membrane species predicted to reside in the fractions analyzed. Equal amounts of total protein were loaded into each lane. All samples were run on the same gel and images shown are derived from the same membrane. For the core cytosolic blot (B, bottom right) exposure levels were increased 4 fold in order to observe the signal. C) The total HCV RNA present in the nuclear, microsomal, MAM, or cytosolic fractions analyzed in panel A was determined by qPCR. The number of infectious HCV particles in these same fractions was determined by infecting Huh7.5 cells with a portion of the sample from each fraction followed by counting focus-forming units identified using indirect immunofluorescence microscopy and antibodies directed against HCV core protein.