Further studies on hepatitis C virus NS5A–SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling

Abstract

The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.

Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon

“…Firstly, we have previously shown that AP-1 inhibition via the Ras-ERK pathway is blocked both by NS5A alone and also by the full-length HCV polyprotein. Secondly, we have recently demonstrated (Macdonald et al, 2005) that AP-1 activation is also inhibited in cells harbouring the FK5.1 replicon and that this block is abrogated by mutation of an SH3 domain-binding polyproline motif in NS5A (a mutation that also abrogates Grb2 binding). One discrepancy that needs to be clarified is the reported ability of the Core protein to stimulate multiple MAPK pathways (Fukuda et al, 2001;Erhardt et al, 2002).…”

Tagging of NS5A expressed from a functional hepatitis C virus replicon

“…First, we needed to confirm that the replicon could tolerate insertions at this location. Therefore, the EGFP-coding region flanked by short hinge regions was amplified by PCR (primer sequences available upon request) and ligated into the BsaBI site of pLRM(wt) (Macdonald et al, 2005), a plasmid containing an NsiI-NsiI fragment (nt 3682-7122) of pFK5.1neo (Krieger et al, 2001). The modified NsiI-NsiI fragment was then reintroduced into pFK5.1neo, generating pFK5.1neo EGFP (Fig.…”

Construction of an infectious cDNA clone of genotype 1 avian hepatitis E virus: characterization of its pathogenicity in broiler breeders and demonstration of its utility in studying the role of the hypervariable region in virus replication

“…The luciferase activity value measured in the HVRd1-luc mutant was almost the same as that of the wild-type replicon statistically. It has been demonstrated that the PxxP motif that binds to the SH3 domain may be involved in enhancing the replication efficiency or infectivity (Bliska, 1996;Macdonald et al, 2005;Pudupakam et al, 2011). A distinctive characteristic of the HVRd1 mutant is the number of proline-rich (PxxP) motifs.…”