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.

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 Introduction 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 [1] . 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] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [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] [21] [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.