Abstract
The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.
Highlights
Hepatitis C virus (HCV) is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses [1]
NS5A is comprised of three domains–we show that the first of these plays a role in the production of new, infectious virus particles
We propose that domain I of NS5A plays multiple roles in virus assembly
Summary
Hepatitis C virus (HCV) is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses [1]. Co- and post-translational proteolytic cleavage of this precursor by cellular and viral enzymes yields the structural proteins: Core, envelope glycoproteins E1 and E2, and the p7 ion channel, which are involved in viral assembly, along with non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. NS proteins are expressed in virus-infected cells but are not incorporated into virus particles; directly involved in RNA synthesis, they play roles in modulation of host defence mechanisms and virus assembly [7,8]. In addition to NS5A, whose roles are detailed below, recent studies have provided evidence for the involvement of NS3, NS4B and NS5B in the later stages of the virus lifecycle–namely virus assembly and release [9,10,11,12,13]
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