Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

Abstract

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

“…We previously assigned the spectra of the D2 and D3 domains of HCV NS5A when isolated in solution (53,56,57,67). To monitor by NMR spectroscopy the effects induced by NS5B ⌬21 on the spectrum of NS5A-D2 (or NS5A-D3), we mixed at equimolar concentrations (i.e.…”

“…Region C is different, as it carries besides the three negatively charged residues also four positively charged residues (four Arg). Although we previously showed that the D2 domain is essentially unfolded and that this property is conserved across HCV genotypes (53)(54)(55)67), we reanalyzed the experimental 13 C NMR chemical shifts corresponding to the C ␣ and C ␤ atoms of NS5A-D2 (Biological Magnetic Resonance Bank accession number 16165 (53)) with the SSP (secondary structure propensities) program (68). This novel algorithm detected a residual secondary structure in the three interaction regions of NS5A-D2 (Fig.…”

“…Assignment of the NS5A-D2, NS5A-D3, and CypA spectra was taken from our previous studies (53,57). Surface Plasmon Resonance Analyses of the NS5B ⌬21 -NS5A-D2, CypA-NS5B ⌬21 , and CypA-NS5A-D2 Interactions-All materials and chemicals came from Biacore (GE Healthcare).…”

“…In previous studies we have shown that both NS5A-D2 (53) and NS5A-D3 (57) establish direct interactions with CypA in vitro and that these domains are substrates for the peptidyl-prolyl cis-trans isomerase activity of CypA. Using NMR spectroscopy, we have mapped onto NS5A-D2 the CypA interactions sites and found a correlation with CsA-resistance mutations or even mutations that impair RNA replication (53).…”

“…It has been shown that NS5A-D1 may adopt two different heterodimeric structures, at least in the context of a crystal (51,52). NS5A-D2 (250 -342), which is essential for RNA replication (47), and NS5A-D3 (356 -447), which is involved in the production and assembly of viral particles (45), have been shown to be natively unfolded (53)(54)(55)(56)(57). In previous studies we have shown that both NS5A-D2 (53) and NS5A-D3 (57) establish direct interactions with CypA in vitro and that these domains are substrates for the peptidyl-prolyl cis-trans isomerase activity of CypA.…”