A conserved tandem cyclophilin-binding site in hepatitis C virus nonstructural protein 5A regulates Alisporivir susceptibility

Abstract

Cyclophilin A (CyPA) and its peptidyl-prolyl isomerase (PPIase) activity play an essential role in hepatitis C virus (HCV) replication, and mounting evidence indicates that nonstructural protein 5A (NS5A) is the major target of CyPA. However, neither a consensus CyPA-binding motif nor specific proline substrates that regulate CyPA dependence and sensitivity to cyclophilin inhibitors (CPIs) have been defined to date. We systematically characterized all proline residues in NS5A domain II, low-complexity sequence II (LCS-II), and domain III with both biochemical binding and functional replication assays. A tandem cyclophilin-binding site spanning domain II and LCS-II was identified. The first site contains a consensus sequence motif of AØPXW (where Ø is a hydrophobic residue) that is highly conserved in the majority of the genotypes of HCV (six of seven; the remaining genotype has VØPXW). The second tandem site contains a similar motif, and the ØP sequence is again conserved in six of the seven genotypes. Consistent with the similarity of their sequences, peptides representing the two binding motifs competed for CyPA binding in a spot-binding assay and induced similar chemical shifts when bound to the active site of CyPA. The two prolines (P310 and P341 of Japanese fulminant hepatitis 1 [JFH-1]) contained in these motifs, as well as a conserved tryptophan in the spacer region, were required for CyPA binding, HCV replication, and CPI resistance. Together, these data provide a high-resolution mapping of proline residues important for CyPA binding and identify critical amino acids modulating HCV susceptibility to the clinical CPI Alisporivir.

Fig 1

CyPA interacts with JFH-1 NS5A peptides in a spot-binding assay. (A) Validation of a spot-binding assay using reported CyPA-binding peptides. Peptides (∼5 nmol) synthesized on a cellulose membrane filter were incubated with HRP-conjugated CyPA (top row), HRP-conjugated R55A mutant CyPA (second row), and CyPA-HRP in the presence of DEB-025 (450 nM) (third row). CyPA-bound spots were identified by exposure to an X-ray film after chemiluminescent substrate incubation. Ponceau S staining was used to demonstrate that the spots for both the wt and the proline mutant (P−) of each sequence contained comparable amounts of peptides. Note that each experiment was done at least twice and wt-CyPA experiments were carried out using regenerated membrane after the R55A and DEB-025 experiments, indicating that the negative results of the R55A and DEB-025 were not because of membrane deterioration. (B) SDS-PAGE image of CyPA and R55A recombinant proteins used in this study to make the HRP conjugates; (C) schematic representation of the 18 overlapping peptides (designated P1 through P18) that span domains II and III of JFH-1 NS5A; (D) CyPA-HRP interacts with immobilized P6 (residues 305 to 322) and P8 (residues 329 to 346) peptides; (E) CyPA binding by P6 and P8 peptides was sensitive to CPI DEB-025 and PPIase active-site mutation R55A; (F) NS5A sequence encompassing P6, P7, and P8 peptides. The D316 Y317 motif that regulates CsA sensitivity is shown in red.

Fig 2

Amino acid residues within the P6 peptide influence CyPA binding. (A) Wild-type (wt), proline-deficient (PA), and single substitutions were evaluated for their ability to bind CyPA; (B) the W312A mutation resulted in reduced replication of a full-length J6-JFH genome. HCV RNAs of wt, W312A, and a polymerase-deficient mutant (GND) were electroporated into Huh-7.5 sh-Luc or sh-A161 stable cell lines. Luciferase assays were performed 4, 24, 48, and 72 h after electroporation. Values were normalized to the 4-h reading, and error bars represent standard deviations from two independent experiments.

Fig 3

CyPA binding to P6 peptide is conserved across all seven genotypes. (A) Amino acid sequences of the P6 and P6′ (sequence shifted one amino acid upstream of P6) peptides of the indicated genotypes (the NCBI accession numbers for the sequences selected are 1a, AAB67036.1; 1b, AJ238799.1; 2a, AB047639.1; 2b, D10988.1; 3a, ADF97231.1; 4a, CAA72338.1; 5a, AAC61696.1; 6a, CAA72801.1; 7a, ABN05226.1). Note the unique N-terminal tyrosine residue in the GT3a P6 sequence. (B) CyPA bound to P6/P6′ of different genotypes in a PPIase-dependent and CPI-sensitive manner. Ponceau S staining revealed that the differences in binding strengths of the different P6/P6′ sequences were not due to the amount of peptides spotted.

Fig 4

P6 and P8 represent a tandem CyPA-binding site of HCV NS5A. (A) Soluble P6, P7, and P8 peptides were evaluated for their ability to compete with NS5A from JFH-1-infected cell lysates for CyPA binding. The indicated peptides and DEB-025 were added to His-CyPA and incubated before NS5A-containing lysate was added to the pulldown assay. (B) Soluble P8 peptides from all seven genotypes were capable of binding to CyPA in the competition assay. Peptides were used at a 3.3-fold molar excess of the His-CyPA. (C) Proline at position 341 is the major contributor to CyPA binding by the JFH-1 P8 peptide. (D) P310 and P341 are the principal CyPA-binding sites on NS5A. HCV RNA containing NS5A of wt, P310A, P341A, P341-343A, or P310A P341A were expressed in Huh-7.5 cells using a recombinant vaccinia virus system and tested in the His-CyPA pulldown assay. The image shown is representative of two independent experiments. Note that in experiments where the two forms of phospho-NS5A (p56 and p58) were resolved by SDS-PAGE, CyPA binding was not biased toward either form, in agreement with previous results (63).

Fig 5

P6 and P8 bind to the ligand-binding pocket of CyPA with similar binding strengths. (A) Soluble P8 peptide competed with P6 for CyPA binding in a proline-dependent manner. Soluble P8/PA8 peptides were included in approximately equal amounts as the total P6 peptides on the membrane (∼5 nmol per spot) in the spot-binding assay that measured P6-CyPA interaction. CyPA binding to P6 spots was reduced by the inclusion of the P8 but not the PA8 peptide. (B) NMR spectra showing the effect of P8 titration on the chemical shifts. Some resonances are insensitive to P8, while others show high sensitivity in both the ¹H and ¹⁵N chemical shifts. (C) Chemical shift changes (Δδ) were quantified and plotted for each residue of CyPA. (D) Residues that exhibited chemical shift perturbations were highlighted in a structure of CyPA complexed with a fragment of the HIV-1 Gag protein (PDB access code 1FGL). Normalized Δδ values for P8 titration (65) were used to generate the color scheme: red, 0.75 < Δδ < 1; pink, 0.5 < Δδ < 0.74; white, Δδ < 0.50. (E) Residues with normalized Δδ values for P6 titration are shown in the same structure of CyPA. The color scheme is the same as that described for panel C.

Fig 6

Alanine scanning of NS5A LCS-II and domain III in the background of DEYN mutations identify P310 and P341 as important for CyPA dependence. Prolines from P341 to P446 were mutated in singlets, doublets, or triplets within the DEYN/J6-JFH construct. Mutant RNAs were electroporated into Huh-7.5 sh-Luc and sh-A161 cell lines. Luciferase assays were performed 4, 24, 48, and 72 h after electroporation. Values were normalized to the 4-h reading in panels A, B, and D, and error bars represent standard deviations from two independent experiments. Solid lines, sh-Luc cells; dashed lines, sh-A161 cells. (A) Replication profiles of proline mutants of LCS-II and domain III. Top, the wt phenotype compared to the DEYN phenotype demonstrates rescue in the sh-A161 cell line; middle left, representative replication kinetics of single mutations (e.g., DEYN P405A); middle right, those of double mutations (e.g., DEYN P417-418A); bottom, those of triple mutations. Whereas the DEYN P349-351A mutant exhibited a phenotype similar to that of the DEYN virus, the DEYN P341-343A mutant uniquely abrogates the ability of DEYN mutations to rescue replication in the sh-A161 cell line (please see the summary of all mutants in Table 4). (B) The individual DEYN P341A, P342A, and P343A mutations highlight the importance of P341 in CyPA dependence. (C) P310A and P341A increase sensitivity to CsA treatment. J6-JFH RNAs with indicated mutations were electroporated into the Huh-7.5 sh-Luc cell line and treated with CsA 24 h after electroporation. Cells were collected 72 h after treatment and assayed for luciferase activity. Fold changes in response to treatment were normalized to no treatment (set at 1) and plotted. (D) DEYN mutations were unable to rescue the lethal phenotype of P310A P341A.

Fig 7

ALPAW-containing peptides from cellular proteins interact with CyPA in a spot assay. A BLAST search of the GenBank database identified two cellular proteins, Supervillin and Nsun5, that contain a linear peptide with the ALPAW motif. The wt peptides, but not the proline-deficient mutants (sequences shown in panel A), bound to CyPA in a PPIase-dependent and DEB-025-sensitive manner (B).