Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication

Abstract

Edited by Charles E. Samuel Implicated in numerous human diseases, intrinsically disordered proteins (IDPs) are dynamic ensembles of interconverting conformers that often contain many proline residues. Whether and how proline conformation regulates the functional aspects of IDPs remains an open question, however. Here, we studied the disordered domain 2 of nonstructural protein 5A (NS5A-D2) of hepatitis C virus (HCV). NS5A-D2 comprises a short structural motif (PW-turn) embedded in a proline-rich sequence, whose interaction with the human prolyl isomerase cyclophilin A (CypA) is essential for viral RNA replication. Using NMR, we show here that the PW-turn motif exists in a conformational equilibrium between folded and disordered states. We found that the fraction of conformers in the NS5A-D2 ensemble that adopt the structured motif is allosterically modulated both by the cis/trans isomerization of the surrounding prolines that are CypA substrates and by substitutions conferring resistance to cyclophilin inhibitor. Moreover, we noted that this fraction is directly correlated with HCV RNA replication efficiency. We conclude that CypA can fine-tune the dynamic ensemble of the disordered NS5A-D2, thereby regulating viral RNA replication efficiency.

In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

“…This displacement is accompanied by a decrease in linewidth and an increase in signal intensity. This indicates that the speed of interconversion between the two states is modified at high pH, and becomes slower in NMR intermediate exchange timescale (μs-ms) [52,53,69], as we have previously suggested based on NMR spin relaxation experiments [31].…”

“…Interestingly, the difference in frequency between these two resonances assigned to the same proton decreased at high temperature, and this relates to an increase in intensity (and decrease in linewidth) for the folded Trp-Hε resonance (Figure 3c, Figure S5). The difference in frequency also decreased at higher pH (Figure 3b), and the resulted resonances at pH 8 and 9 fell in a linear chemical shift pattern defined by the two above mentioned unfolded and folded Trp-Hε resonances [52,53]. This behavior is strongly indicative of a chemical exchange in the intermediate timescale (μs-100 ms) between a disordered and a folded conformation, that becomes faster at higher temperature and at higher pH [54].…”

Cyclophilins and Their Roles in Hepatitis C Virus and Flavivirus Infections: Perspectives for Novel Antiviral Approaches

“…The binding of CypA to a proline-rich region motif in NS5A-D2 enhances its RNA-binding properties [73]. This was corroborated by Dujardin et al in a recent study where CypA was shown to allosterically modulate the disordered NS5A-D2, regulating viral RNA replication efficiency [78]. CypA and NS5B share overlapping binding sites in NS5A-D2 [79], and Ngure et al showed that the RNA-binding region in NS5A-D2 coincides with the binding site of CypA and NS5B [80], suggesting the formation of a ternary complex between CypA, NS5A and NS5B that regulates viral RNA replication [81].…”

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts

“…This displacement is accompanied by a decrease in linewidth and an increase in signal intensity. This indicates that the speed of interconversion between the two states is modified at high pH and becomes slower in NMR intermediate exchange timescale (μs-ms) [ 52 , 53 , 69 ], as we have previously suggested based on NMR spin relaxation experiments [ 31 ].…”

“…Interestingly, the difference in frequency between these two resonances assigned to the same proton decreased at high temperature, and this relates to an increase in intensity (and decrease in linewidth) for the folded Trp-Hε resonance ( Figure 3 c, Figure S5 ). The difference in frequency also decreased at higher pH ( Figure 3 b), and the resulting resonances at pH 8 and 9 fell in a linear chemical shift pattern defined by the two above-mentioned unfolded and folded Trp-Hε resonances [ 52 , 53 ]. This behavior is strongly indicative of a chemical exchange between a disordered and a folded conformation.…”