A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B

Abstract

GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras-Erk signalling, however GBV-B NS5A expression did result in the elevation of b-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras-Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses. INTRODUCTIONGB virus B (GBV-B) is the closest phylogenetic relative of hepatitis C virus (HCV) (Muerhoff et al., 1995;Ohba et al., 1996); it shares up to 28 % overall amino acid identity with HCV, rising to 50 % conservation in regions such as the NS3 coding sequence. GBV-B has been proposed as a model for HCV infection and has been used to characterize candidate antivirals as well as for pathogenesis studies (Beames et al., 2001;Bright et al., 2004). It shares the hepatotropism of HCV. Although GBV-B has been reported to lead to persistent infections in some cases in tamarins (Saguinus species) (Martin et al., 2003;Nam et al., 2004), unlike the human virus, GBV-B primarily causes an acute, resolving hepatitis in tamarins and other New World monkeys such as common marmosets (Callithrix jacchus) (Bukh et al., 2001;Jacob et al., 2004;Lanford et al., 2003). In contrast, HCV is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, affecting an estimated 3 % of the world's population.Both GBV-B and HCV are members of the genus Hepacivirus of the family Flaviviridae and have a positivesense RNA genome that is translated in a cap-independent fashion to generate a polyprotein, cleaved by host and viral proteases to produce 10 mature viral proteins. There are three structural proteins at the N terminus: core, E1 and E2, followed by a small cation channel, p7 (p13 in GBV-B). The C-terminal t...

Modeling HCV disease in animals: virology, immunology and pathogenesis of HCV and GBV-B infections

“…Even the differential infection profiles between the two viruses could prove helpful in grasping the immune evasion strategies of HCV. For example, the HCV non-structural protein 5A (NS5A), implicated in establishing persistence, modulates the Ras-Erk signaling pathway ( Mankouri et al, 2008a ), whereas the GBV-B NS5A protein does not block this pathway and also differs in its cellular distribution ( Mankouri et al, 2008b ). This suggests that the altered mechanism of NS5A in HCV infection could be responsible for the chronicity of HCV in humans.…”

“…At a functional level, the NS3 protease of GBV-B can correctly process the HCV polyprotein ( Scarselli et al, 1997 ) and HCV/GBVB chimeric NS3 proteins are enzymatically active ( Butkiewicz et al, 2000 ). Interestingly, while both HCV and GBV-B NS5A proteins activate the p13 kinase pathway, HCV NS5A blocks the Ras-Erk pathway, whereas GBV-B NS5A does not ( Macdonald et al, 2003 , 2004 ; Mankouri et al, 2008b ). Despite an error-prone RdRp, very few amino acid substitutions have been observed, limiting the quasispecies variation of GBV-B in infected animals ( McGarvey et al, 2008 ).…”

A Cooperative Interaction between Nontranslated RNA Sequences and NS5A Protein Promotes In Vivo Fitness of a Chimeric Hepatitis C/GB Virus B

“…We and others have shown that GBV-B shares a number of features with HCV, but also exhibits some differences, in terms of genomic organization [8], [9], viral protein maturation [10]–[12], and virus/host interactions [13]–[16]. The GBV-B genome contains a single open reading frame encoding a polyprotein which is subsequently cleaved into three structural proteins and seven nonstructural (NS) proteins, among which NS3 to NS5B, only, are required for efficient genome replication in cell culture [17].…”

Nonstructural 5A (NS5A) protein of hepatitis C virus : self-association, membrane anchoring and interactions with antivirals

“…We performed a bioinformatics analysis of NS5A protein sequence to understand variations within the protein from different genotypes of HCV. NS5A is a unique protein that has no human or viral homologs other than NS5A of GB virus B (GBV-B) with 21% identity (Mankouri et al, 2008). Herein, UniProt database was used to retrieve NS5A protein sequences from the different genotypes of HCV (i.e.…”

“…Neither NS5A full-length protein nor NS5A-DI has any enzymatic function that can be tested in vitro (Mankouri et al, 2008). However, the DI is known to be interacting with the RNA and DCV (Gao et al, 2010;Hwang et al, 2010).…”