Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions

Abstract

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

A pivotal role of serine 225 phosphorylation in the function of hepatitis C virus NS5A revealed with the application of a phosphopeptide antiserum and super-resolution microscopy

“…We also used confocal microscopy with Airyscan to analyse the distribution of both total NS5A and pS225-NS5A in cells infected with the various phosphoablatant mutants. As shown in Fig 6, all mutants displayed a broad cytoplasmic distribution of total NS5A that was similar to wildtype, with the exception of S225A which exhibited a more compact perinuclear distribution, as documented previously (23). However, as observed in Fig 1, we noted again that in all cases there was a subset of total NS5A reactivity that did not stain for pS225, and this led us to investigate this observation in more detail.…”

“…We previously demonstrated a role for S225 phosphorylation in HCV genome replication, and further showed that this was likely mediated via interactions of NS5A with a defined subset of cellular factors (23, 37). A caveat to these studies was the necessary dependence on interpretation of data based on the analysis of the phenotype of S225 mutants (S225A phosphoablatant or S225D phosphomimetic).…”

“…NS5A is phosphorylated on multiple serine and (to a lesser extent) threonine residues (28). Whereas p56 is reportedly phosphorylated within DII and DIII, in contrast, multiple phosphorylation events within the serine-rich LCSI result in the production of p58 (Fig 1A) (23, 29-31).…”

“…NS5A has been shown to associate with lipid droplets (LDs) in infected cells (22), where it is postulated to physically link the processes of genome replication and virus assembly (19). NS5A also interacts with many cellular proteins (23, 24) and signalling pathways (25-27), modifying the host cell environment to favour the virus, this may require a subset of NS5A distinct from that involved in genome replication or virus assembly.…”

“…Our previous studies had pointed to a key role for S225 phosphorylation – the S225A phosphoablatant mutant had a dramatic phenotype: a loss of p58, a 10-fold reduction in JFH-1 genome replication, a lack of interactions between NS5A and a range of host proteins, and a perinuclear restricted localisation of NS5A and other replication complex components (23, 37). This phenotype prompted us to investigate the role of S225 phosphorylation more directly, therefore to complement the analysis of S225 mutants, we raised an antiserum to pS225-NS5A and used this unique reagent to probe the functions of S225-phosphorylated wildtype NS5A.…”