The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.
Author Summary Hepatitis C virus (HCV) is a positive-strand RNA virus and it is a major cause of liver disease worldwide affecting more than 170 million individuals. Infection of cells with HCV leads to rearrangement of cytoplasmic host cell membranes and the formation of the membranous web (MW) containing viral replication and assembly complexes. The MW is thought to function in concentrating viral components, regulating virus replication, and immune evasion. Our analysis has provided new insight into the organization of the MW and the mechanisms that contribute to the formation and maintenance of distinct compartments within the MW. We show that the MW limits access of host cell innate immune receptors to sites of viral replication and assembly. Moreover, we show that components of the nuclear transport machinery, normally involved in regulating traffic between the cytoplasm and the nucleus, have a role in limiting immune receptor access to compartments within the MW. These findings provide important insights in how HCV, and likely other positive-strand RNA viruses, organize their replication factories and evaded recognition by host cell immune receptors.