Abstract
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.
Highlights
Positive-strand RNA viruses account for a significant portion of the total viral diseases affecting humans around the world
The barrier formed by the membrane structures of the membranous web (MW) appears to be selectively permeable, and the nuclear transport machinery contributes to controlling access of proteins to compartments within the MW [37, 38]
In Hepatitis C virus (HCV)-infected cells, RIG-I and MDA5 were absent from larger regions of the cytoplasm and, importantly, these areas contained the majority of HCV proteins examined and viral doublestranded RNA (dsRNA) (Fig 1A and 1B bottom panels)
Summary
Positive-strand RNA viruses account for a significant portion of the total viral diseases affecting humans around the world Within this class of viruses is the Flaviviridae family, consisting of four viral genera, including Flavivirus and Hepacivirus. Host cell membrane rearrangements have been observed for all positive-strand RNA viruses and they can generally be characterized by the induction of two different membrane alterations: those containing double membrane vesicles, and those that form invaginated vesicles or spherules [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19]. The precise role of DMVs in the viral life cycle and the spatial organization of different viral processes within the MW have not yet been described
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