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.

How replication/assembly sites in positive-strand RNA virus-infected cells establish, compartmentalize, and segregate various biochemical activities remains largely ill defined. Within the complex architecture of the MW, it has been suggested that positive-strand RNA viruses coordinate different viral processes, such as replication and assembly, by spatially segregating these processes in different compartments [48, 57, 58] . Current theories on HCV assembly and egress propose that HCV RNA moves with the core protein from replication compartments to sites of virus assembly, suggesting a spatial separation between these processes [64, 65] . This idea is supported by several previous studies which indicate that viral maturation and assembly is mediated by the trafficking of proteins, including the NS2/p7 complex and the NS3/4A complex, from replication compartments to lipid droplets, which initiates assembly of viral particles [52, [66] [67] [68] [69] [70] [71] . However, visualization and characterization of these different compartments has been difficult. In this study, we used subcellular fractionation techniques to isolate membrane fractions that were enriched for either viral replication or assembly components (Fig 4) . The predominance of the viral polymerase NS5B in microsome-containing fractions implies an enrichment of replication complexes, while the concentration of the core protein in a membrane fraction that has similar sedimentation and buoyant density characteristics to MAMs suggests that this membrane fraction is enriched for assembly complexes. Consistent with these observations, increased levels of viral RNA co-fractionate with the microsomal fraction while increased levels of infectious virus are associated with the MAM membrane fraction ( Fig 4B) . Further analysis of the subcellular fractions also revealed that host proteins which function in replication (PI4K) were found in the microsomal fraction and those involved in assembly (ApoE) were present in the MAM fraction (Fig 4C) . These results support the existence of distinct membrane compartments contributing to the bulk of replication and assembly and show that subcellular fractionation can be used to separate and characterize these different viral compartments.