Hepatitis C virus NS5A protein blocks epidermal growth factor receptor degradation via a proline motif- dependent interaction.

Zsofia Igloi,Kalle Saksela,Andrew Macdonald,Mark Harris,Jamel Mankouri,Arunas Kazlauskas

doi:10.1099/vir.0.000145

Abstract

Hepatitis C virus (HCV) establishes a persistent infection that in many cases leads to cirrhosis and hepatocellular carcinoma. The non-structural 5A protein (NS5A) has been implicated in this process as it contains a C-terminal polyproline motif (termed P2) that binds to Src homology 3 (SH3) domains to regulate cellular signalling and trafficking pathways. We have shown previously that NS5A impaired epidermal growth factor (EGF) receptor (EGFR) endocytosis, thereby inhibiting EGF-stimulated EGFR degradation by a mechanism that remained unclear. As EGFR has been implicated in HCV cell entry and trafficking of the receptor involves several SH3-domain containing proteins, we investigated in more detail the mechanisms by which NS5A perturbs EGFR trafficking. We demonstrated that the P2 motif was required for the NS5A-mediated disruption to EGFR trafficking. We further demonstrated that the P2 motif was required for an interaction between NS5A and CMS, a homologue of CIN85 that has previously been implicated in EGFR endocytosis. We provided evidence that CMS was involved in the NS5A-mediated perturbation of EGFR trafficking. We also showed that NS5A effected a loss of EGFR ubiquitination in a P2-motif-dependent fashion. These data provide clues to the mechanism by which NS5A regulates the trafficking of a key cellular receptor and demonstrate for the first time the ability of NS5A to regulate host cell ubiquitination pathways.

Highlights

Hepatitis C virus (HCV) is an enveloped virus with a positive-sense, ssRNA genome belonging to the genus Hepacivirus within the family Flaviviridae
We have previously shown that non-structural 5A protein (NS5A) partially localized to early endosomes and, it had no effect on epidermal growth factor (EGF) internalization, colocalized with the epidermal growth factor receptor (EGFR) and altered its distribution (Mankouri et al, 2008)
As we have previously shown that this motif was not required for virus replication http://vir.sgmjournals.org (Hughes et al, 2009b), we were able to evaluate the cellular functions of the P2 motif in Huh7 cells either stably harbouring subgenomic replicons (SGRs) or infected with JFH-1 virus in which the motif had been disrupted by alanine substitution (PA2)

Summary

Introduction

Hepatitis C virus (HCV) is an enveloped virus with a positive-sense, ssRNA genome belonging to the genus Hepacivirus within the family Flaviviridae. The 9.6 kb HCV genome encodes a single large polyprotein of 3000 aa which is processed co- and post-translationally by viral and host cellular proteases into 10 functional proteins These include three structural proteins (core, E1 and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Scheel & Rice, 2013). The LCS between domains II and III is proline-rich (Fig. 1a) and, of note, contains a highly conserved PxxPxR sequence (Macdonald et al, 2004, 2005b; Tan et al, 1999) This sequence matches the consensus for a class II SH3-domain-binding motif (Mayer, 2001) and is referred to as the P2 motif. Several cellular SH3domain-containing binding partners for the P2 motif have been identified (Macdonald et al, 2004; Nanda et al, 2006), but their roles in the HCV life cycle remain undefined

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Results

Conclusion