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

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.

In both models, the K/W4 side chain does not have access to the pore lumen but is accessible at the protein surface on the cytosolic side at the membrane interface. W4 could thus slightly disturb interaction(s) with p7 partner(s).
In both models, the L/W5 side chain could point to the pore lumen and thus could play a role in modulating ion flux and/or in ion selectivity. Model 1 OuYang et al. (2013) In model 2, residue 6 is involved in helix-helix interactions. The W6 side chain could disturb the folding/stability of the N-terminal six-helix bundle and thus disturb ion channel activity.

W6 W6
In model 1, the W6 side chain occupies the pore lumen and thus could disturb/prevent ion flux. In both models, the I/W7 side chain does not access the pore lumen but is accessible at the protein surface on the cytosolic side at the membrane interface. Both Trp and Ile are hydrophobic residues that could play a similar role in subunit interactions in hexamer assembly and interaction with membrane lipids. The enhanced effect of I7W on virus production could be due to a stabilization of the folding/assembly of the N-and/or C-terminal parts of p7. In both models, the L/W7 side chain does not access the pore and is poorly accessible at the protein surface. Both Trp and Leu are hydrophobic residues that could play a similar role in subunit interactions in hexamer assembly. The effect of I7W on virus production could be due to a perturbation of the folding/assembly of the N-and/or C-terminal parts of p7. In both models, the W10 side chain does not access the pore lumen. In model 2, the W10 side chain is accessible at the protein surface and thus could disturb interaction(s) with p7 partner(s). This hypothesis might be also valid for model 1, although the accessibility of W10 is limited at the surface of this model. Because the W10 side chain interacts with many residues of neighboring subunits in model 1, an alternative explanation would be that this mutation rigidifies the oligomeric structure and thus prevents conformational changes that are required for pore functioning.  In both models, the A/W11 side chain is accessible at the protein surface and is close to residues of the C-terminus of p7. The A11W mutation could stabilize interaction(s) with p7 partner(s), either directly and/or by stabilizing the folding/assembly of the C-terminal region of p7.

W13 W13
In both models, the side chain of amino acid 13 partially points to the pore and could be involved in p7 subunit interactions. The absence of an effect of this mutation in modulating ion flux is questionable. In both models, the A/W14 side chain does not access the pore but is accessible at the protein surface at the level of the membrane hydrophobic core. Despite its bulky side chain, W14 has no major impact on p7 structure and assembly, likely explaining the absence of any detrimental effect on virus production. In both models, the side chain of amino acid 15 does not access the pore lumen but is accessible at the protein surface and thus might disturb some interaction(s) with p7 partner(s). However, because the W15 side chain interacts with C-terminal residues of neighboring subunits in both models, an alternative explanation would be that this mutation disturbs the folding and/or rigidifies the oligomeric structure and thus prevents conformational changes required for pore functioning.  In model 2, the C/W16 side chain does not access the pore lumen and its accessibility to the protein surface is limited. This position at the subunit interface could be involved in the stability of p7 assembly.

W16 W16
In model 1, the C/W16 side chain is in the pore lumen. It is thus surprising that mutation C16W would have no effect on the ion flux in this model. In model 1, the N/W17 side chain does not access the pore lumen and is accessible at the protein surface at the level of the membrane hydrophobic core. The N17W mutation might thus alter some interactions with p7 partner(s).

W17 W17
In model 2, the N/W17 side chain is in the pore lumen, indicating that the N17W mutation could seriously disturb the ion flux.

W22 W22
In model 1, the F/W22 side chain is located at the subunit interface. The increased virus production observed with F22W mutation could be linked to a stabilization of p7 oligomer assembly.

W23 W23
In model 1, the V/W23 side chain is located at the subunit interface and likely involved in p7 oligomer assembly. Its access to the protein surface is very limited. In both models, residue F26 does not access the pore lumen but is involved in numerous contacts within p7. Thus, it is likely to play a major role in protein assembly. The mutation F26W could disturb the stability of this assembly and/or the functional dynamics of this assembly, leading to a malfunctioning of p7 ion channel. In addition, the Trp side chain is more accessible to the protein surface than that of Phe, and could thus disturb some interactions with p7-interacting partners.

W27
In model 1, the V/W27 side chain can access both the pore lumen and subunit interface. This ambivalence could explain the absence of effect of V27W mutation (although a modulation of ion flux would be expected).
In model 2, the V/W27 side chain is at the subunit interface and is partly accessible at the protein surface at the level of the membrane hydrophobic core. The lack of an effect of this mutation suggests this residue is not essential for interaction with p7 partner(s).

W29 W29
In both models, the A/W29 side chain is located at the subunit interface but also has limited access to the pore lumen, indicating that this residue could modulate ion flux. For the A29W mutant, the bulky side chain of Trp at the subunit interface might facilitate the open state of the pore, allowing an increase of p7 activity.