Vps4 and the ESCRT-III complex are required for the release of infectious hepatitis C virus particles

Abstract

The mechanisms by which infectious hepatitis C virus (HCV) particles are assembled and released from infected cells remain poorly characterized. In this regard, many other enveloped viruses, notably human immunodeficiency virus type 1, have been shown to utilize the host vacuolar protein sorting machinery (also known as the endosomal sorting complex required for transport; ESCRT) to traffic through the cell and effect the membrane rearrangements required for the formation of enveloped particles. We postulated that this might also apply to HCV. To test this hypothesis, we established a method of conditional virus-like particle assembly involving transcomplementation of an envelope-deleted JFH-1 genome using plasmid transfection. This system reliably produced virus particles that were infectious and could be enumerated easily by focusforming assay in Huh7 cells. Following co-transfection with plasmids expressing various dominant-negative forms of either components of the ESCRT-III complex or Vps4 (the AAA ATPase that recycles the ESCRT complexes), a reduction in particle production was seen. No significant effect was observed after co-transfection of dominant-negative ESCRT-I or Alix, an ESCRT associated protein. Dominant-negative Vps4 or ESCRT-III components had no effect on either virus genome replication or the accumulation of intracellular infectious particles. These data were confirmed using cell culture infectious HCV and we conclude that HCV requires late components of the ESCRT pathway for release of infectious virus particles. INTRODUCTIONHepatitis C virus (HCV) represents a major global health burden. An estimated 170 million people are chronically infected worldwide and a significant proportion of these will go on to develop end stage liver disease. Current drug treatments are poorly tolerated and are only effective in approximately 50 % of individuals. There is, therefore, a pressing need for a greater understanding of the molecular mechanisms involved in HCV infection in order for novel drug targets to be identified.HCV is the prototype member of the genus Hepacivirus in the family Flaviviridae. It is a single-stranded, positive-sense RNA virus whose genome encodes a single ORF. This is translated in a cap-independent manner from an internal ribosome entry site (IRES) located in the 59 untranslated region, into a single polyprotein of approximately 3000 aa. The polyprotein is proteolytically cleaved by host and viral proteases to form 10 mature proteins; the structural proteins (core, E1 and E2), a viroporin p7 and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B).Although recent advances in the development of tissue culture systems for the study of the HCV life cycle (Lindenbach et al., 2005;Wakita et al., 2005;Zhong et al., 2005) have defined a critical role for lipid droplets in virus assembly (Miyanari et al., 2007), the precise mechanism by which infectious HCV particles are assembled and released remains poorly characterized. In particular, it has not been established how nascent...

The PSAP Motif within the ORF3 Protein of an Avian Strain of the Hepatitis E Virus Is Not Critical for Viral InfectivityIn Vivobut Plays a Role in Virus Release

“…Numerous RNA viruses, including retroviruses, rhabdoviruses, filoviruses, flaviviruses, orbiviruses, orthomyxoviruses, and arenaviruses (8,12,16,25,47,52,61) use the ESCRT pathway to release virus particles from the cells. Recent work suggests that a PXXP motif within the HEV ORF3 protein may play a similar role during HEV particle formation (10,45,46,62).…”

Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

“…Thereafter, budding occurs concurrently with the release of ESCRT components from the MVB membrane induced by an AAA-ATPase, Vps4, which is present in humans as two isoforms, Vps4A and Vps4B (Babst et al, 1998;Katzmann et al, 2002). Numerous enveloped RNA viruses such as human immunodeficiency virus type 1 (HIV-1) (Garrus et al, 2001), Ebola virus (Martin-Serrano et al, 2001) and avian sarcoma virus (ASV) (Pincetic et al, 2008) and, more recently, hepatitis C virus (HCV) (Ariumi et al, 2011;Corless et al, 2010;Lai et al, 2010) have been shown to utilize ESCRT complexes during virion morphogenesis. Furthermore, enveloped DNA viruses including hepatitis B virus (HBV) (Lambert et al, 2007;Watanabe et al, 2007) and herpes simplex virus 1 (HSV-1) (Crump et al, 2007) have been reported to exploit the MVB machinery.…”

The Roles of ESCRT Proteins in Healthy Cells and in Disease

“…Both ESCRT-I and Alix can independently recruit ESCRT-III, which together with Vps4 are required for efficient virus budding. Recent studies have shown that ESCRT-III and Vps4 can be recruited independently of either Tsg101 or Alix by the herpes simplex virus type-1 (Pawliczek & Crump et al, 2009) and the hepatitis C virus (Corless et al, 2010). ESCRT-II was found not to be essential for HIV-budding (Langelier et al, 2006), however ESCRT-II was discovered recently to be essential for release of the avian sarcoma virus (Pincetic et al, 2008).…”