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.

Our localization studies detect the presence of viral RNA in distinct locations in the cell, primarily within the MW but also in the surrounding cytoplasm. This distribution is consistent with the various pathways in which both positive-and negative-strand viral RNA are used, including genome replication, translation, and viral assembly, and the proposed spatial separation for these processes in cells infected with HCV or various other positive-strand RNA viruses [11, 24, 52] . To further evaluate the distribution of key HCV proteins and RNA in cells, we have used subcellular fractionation to isolate various membrane fractions from infected cells. The bulk of HCV proteins examined were contained within two cytoplasmic membrane fractions. One lighter membrane fraction contains all the cytoplasmic membranes that did not sediment into a mitochondrial pellet fraction, including ER-derived microsomal membranes, which we term the microsomal fraction. The second contains membranes associated with the mitochondrial pellet including a mitochondrial-associated membranes (MAMs) and cytoplasmic membranes with similar sedimentation and buoyant density characteristics. We will refer to this fraction as the MAM fraction. In HCV-infected cells, both of the 'microsomal' and 'MAM' fractions are predicted to contain membranes with unique characteristics arising during the HCV-induced changes in cellular membrane structure.