The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

<div><p>The hepatitis C virus (HCV) p7 protein is required for infectious virus production via its role in assembly and ion channel activity. Although NMR structures of p7 have been reported, the location of secondary structural elements and orientation of the p7 transmembrane domains differ among models. Furthermore, the p7 structure-function relationship remains unclear. Here, extensive mutagenesis, coupled with infectious virus production phenotyping and molecular modeling, demonstrates that the N-terminal helical region plays a previously underappreciated yet critical functional role, especially with respect to E2/p7 cleavage efficiency. Interrogation of specific N-terminal helix residues identified as having p7-specific defects and predicted to point toward the channel pore, in a context of independent E2/p7 cleavage, further supports p7 as a structurally plastic, minimalist ion channel. Together, our findings indicate that the p7 N-terminal helical region is critical for E2/p7 processing, protein-protein interactions, ion channel activity, and infectious HCV production.</p></div

Mutation of the N-terminal helix is deleterious for infectious virus production <i>in vitro</i>.

<p><b>A)</b> Cartoon of the N-terminal tryptophan substitutions generated in J6/JFH p7. As in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.g001" target="_blank">Fig 1</a>, the secondary structure boundaries shown were previously deduced from p7 NMR data using HCV-J (genotype 1b) in 50% TFE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref041" target="_blank">41</a>]. <b>B)</b> HCV RNA levels in Huh-7.5 cells 8 and 72 hpe demonstrating that all p7 mutants replicate efficiently. J6/JFH lacking either p7 (Δp7) or the HCV glycoproteins (ΔE1E2) were used as additional assembly-defective controls. <b>C)</b> Infectious virus production quantified by limiting dilution assay on naïve Huh-7.5 cells shows that mutation of various residues in this region preclude generation of infectious HCV particles. <b>D)</b> Western blot analyses detecting HCV E2 antigen. Left panel: J6/JFH WT-, ΔE1E2-, E2-IRES-p7-, or p7-IRES-NS2-replicating Huh-7.5 cell lysates, used here to provide markers for E2p7NS2, E2p7, and E2 protein species. Right panel: Parallel western blot analysis comparing amounts of E2 relative to E2p7 between J6/JFH WT and N-terminal helix p7 mutants (positions 1–13).</p

Amino acid requirements at positions 6, 9, and 12.

<p>Infectious virus in the supernatants of monocistronic <b>(A, B & C)</b> or bicistronic <b>(D, E & F)</b> p7 mutants quantified by limiting dilution assay on naïve Huh-7.5 cells. Mutant viruses yielding significantly less infectious virus production as compared with wild-type were identified using unpaired t-tests. Statistical results are indicated as follows: * p<0.05, ** p<0.01 and *** p<0.001. Black diamond (◆) indicates that these control viruses are monocistronic. Note that the WT, GNN, Δp7, and ΔE1E2 titers reported in panels <b>E</b> and <b>F</b> are duplicated from panel <b>D</b> as these viruses were all analyzed in parallel.</p

Identification of putative ion channel defective mutants by homology modeling and bafilomycin A1 rescue.

<p><b>A)</b> Molecular models of N-terminal region mutants that yielded >1 log reduction in infectious virus production compared to wild-type in a bicistronic context. The mutated residue Trp side chains are shown in red. Models provide insight into whether the mutation is likely to block the pore (e.g. H9W and S12W), disturb p7 intramolecular interactions (e.g. A10W), or interrupt p7 interactions with binding partners (e.g. A1W, A10W). <b>B)</b> Bafilomycin A1 rescue experiment schematic. Forty-eight hours post-electroporation, Huh-7.5 cells replicating control or p7 mutant viruses were supplied with cell culture medium containing bafilomycin A1 [8nM] or DMSO. Supernatants were collected 24 hours post-treatment, concentrated and dialyzed to remove excess bafilomycin A1, and then tittered on naïve Huh-7.5 cells to quantify infectious virus production. <b>C)</b> Resulting infectious virus titers from the experiment outlined in panel b. Mutant viruses yielding significantly more infectious virus production under bafilomycin A1 conditions compared with DMSO were identified using unpaired t-tests. Statistical results are indicated as follows: ns = not significant, * p<0.05, and *** p<0.0001. LOQ: lower limit of the limiting dilution assay. Black diamond (◆) indicates that these control viruses are monocistronic; all others shown are bicistronic. KRAA denotes J6/JFH with K33A and R35A mutations in p7.</p

Passage of deleterious N-terminal mutants identifies second site pseudo-revertants.

<p><b>A)</b> Coding changes identified within p7 by sequencing after passage of original p7 mutant genomes in Huh-7.5 cells. No virus was obtained after passage of H9W (indicated by the diamond). <b>B)</b> Western blot analyses detecting HCV E2 antigen. Left panel: J6/JFH WT-, ΔE1E2-, E2-IRES-p7- or p7-IRES-NS2-replicating Huh-7.5 cell lysates, used here to provide markers for E2p7NS2, E2p7, and E2 protein species. Right panel: Parallel western blot analysis comparing amounts of E2 relative to E2p7 between J6/JFH WT, original p7 mutant genomes and genomes harboring mutations identified after passage. <b>C)</b> HCV RNA levels in Huh-7.5 cells determined 8 and 72 hpe showing all p7 mutants (original and those harboring mutations identified after passage re-engineered into J6/JFH p7) replicate efficiently. <b>D)</b> Infectious virus in the supernatants quantified by limiting dilution assay on naïve Huh-7.5 cells demonstrating rescue of infectivity by mutations identified after passage re-engineered into the J6/JFH backbone.</p

Comparison of p7 structure models identifies the N-terminal helical region as a potential key modulator of p7 function.

<p><b>A)</b> Comparison of amino acid sequences and NMR secondary structural elements of p7 as determined by NMR in 50% TFE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref041" target="_blank">41</a>] (PDB entry, 2K8J), 125 mM DHPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref022" target="_blank">22</a>] (PDB entry, 2MTS), 100% MeOH [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref042" target="_blank">42</a>] (PDB entry, 3ZD0) and 200 mM DPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref047" target="_blank">47</a>] (PDB entry, 2M6X). The stars (*) indicate that additional amino acids (N-terminal FLAG tag and C-terminal polylinker) were fused to this construct. The green box highlights a region where secondary structural elements are quite divergent across models, especially between monomeric (first three sequences) and hexameric (bottom sequence) NMR-based models. h, helix; c, coil; t, turn. The sequence of the p7 (J6) used in this study is also shown for comparison. An amino acid similarity index is used where <i>asterisk</i> indicates invariant, <i>colon</i>, highly similar and <i>dot</i>, similar. <b>B)</b> Comparison of the NMR model in DPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref047" target="_blank">47</a>] and NMR/MD model in POPC [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref044" target="_blank">44</a>] showing the hexameric form and two opposing subunits in the hexamer. Lines shown in the left hand panels represent the membrane interfaces and hydrophobic core (between the middle two lines). The positions of both models relative to the membrane bilayer were deduced from MD simulations in a POPC bilayer as previously reported for model 1 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref053" target="_blank">53</a>] and model 2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref044" target="_blank">44</a>]. N- and C-termini are noted by “N” and “C”, respectively. <b>C)</b> N-terminal helical packing in both models demonstrates similar packing and residues 9 and 12 in both models point towards the pore. Figures were generated from structure coordinates by using VMD (<a href="http://www.ks.uiuc.edu/Research/vmd/" target="_blank">http://www.ks.uiuc.edu/Research/vmd/</a> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005297#ppat.1005297.ref054" target="_blank">54</a>]) and rendered with POV-Ray (<a href="http://www.povray.org/" target="_blank">http://www.povray.org/</a>).</p