Date Published: February 10, 2016
Publisher: Public Library of Science
Author(s): Christopher J. Neufeldt, Michael A. Joyce, Nicholas Van Buuren, Aviad Levin, Karla Kirkegaard, Michael Gale Jr., D. Lorne J. Tyrrell, Richard W. Wozniak, Richard J. Kuhn.
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.
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. HCV is a Hepacivirus that is estimated to infect 170 million people world-wide, and, without treatment, this virus leads to end stage liver disease in approximately 30% of patients . The replication cycle of HCV occurs primarily in the cytoplasm of host cells where, upon entry, the viral genome is translated on the rough endoplasmic reticulum (ER). The resulting HCV polyprotein is then cleaved by both viral and host factors to form 10 distinct proteins. Expression of HCV proteins causes major rearrangements of host cell membranes, leading to the formation of a complex membranous environment conducive to viral replication and assembly, termed the membranous web (MW). The virus-induced MW is essential for the viral replication cycle and harbours compartments that are physically separated from the surrounding cytosol [2, 3]. 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 [4–19]. Replication complexes formed by several flaviviruses, including Dengue virus (DENV) and West Nile virus (WNV), contain ER-derived membrane sheets with numerous invaginated vesicles that maintain contact with the surrounding cytosol through narrow 11 nm pores located at the neck of the vesicle [11, 12]. By contrast, the HCV-induced MW is characterized by the clustering of single membrane vesicles and double membrane vesicles (DMVs) as well as multivesicular bodies, all within specific cytoplasmic regions that are also enriched for lipid droplets and ER membranes [8, 20–22]. Although the architecture and topology of the MW has been extensively studied, the spatial organization and function of its various membrane structures is still poorly understood. Several recent studies have proposed a prominent role for DMVs during HCV infection, by demonstrating that viral replication occurs in association with DMVs, and that these structures are vital for the viral life cycle [8, 23]. However, 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.
Much like other membrane-bound organelles, the membrane structures induced by positive-strand RNA viruses serve to both concentrate proteins within a specific area, thereby increasing efficiency of certain processes, and to spatially separate competitive reactions. A proposed function of sequestering viral replication complexes away from the surrounding cytosol is the concealment of viral PAMPs from RLRs, a process that is predicted to attenuate host cell innate immune activation. On the basis of previous data and that presented here, we propose that structural features of the MW, including components of the nuclear transport machinery, establish a selective permeability barrier between the surrounding cytosol and viral replication and assembly centers within the MW [8, 23, 37, 38, 57]. We show here that this barrier facilitates viral infection by inhibiting the access of RLRs to regions within the MW. These conclusions are supported by our findings that the addition of NLS sequences to either RIG-I or MDA5, which allows these proteins to interface with the nuclear transport machinery, overcome the barrier that restricts their access to compartments within the MW. A consequence of NLS-mediated movement of RLRs into the MW is increased immune activation and the inhibition of viral replication.