Date Published: November 20, 2015
Publisher: Public Library of Science
Author(s): Margaret A. Scull, William M. Schneider, Brenna R. Flatley, Robert Hayden, Canny Fung, Christopher T. Jones, Marieke van de Belt, François Penin, Charles M. Rice, Glenn Randall.
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.
Over 130 million people worldwide are at risk for liver fibrosis, cirrhosis, hepatocellular carcinoma, and end stage liver disease as a result of hepatitis C virus (HCV) infection . These complications of infection have made hepatitis C the most common indication for liver transplantation . Further, while novel direct-acting antivirals targeting HCV have dramatically improved clinical outcomes, no vaccine exists to date, and the disease burden is expected to increase over the next decade .
In this report, we have extensively interrogated the HCV p7 protein via mutagenesis and determined the effects of these mutations on virus replication and infection in cell culture. In addition, we have modeled these mutations using p7 structure information based on previous NMR experiments. Our data confirm previous reports that p7 is not required for viral replication, as all p7 mutants tested replicated with wild-type efficiency. Importantly, our large-scale, structure-function analyses illustrate a global tolerance for amino acid sequence alterations, either by insertion or individual amino acid substitution in the J6/JFH background. These results underscore the structural flexibility of p7  that has been similarly described for other viroporins such as HIV-1 Vpu . Our data are further in line with the conservation of p7 amino acid physico-chemical properties and hydropathic character but not precise sequence across genotypes . Notably, two of the nine conserved amino acids (G18 and Y42) were directly assessed in this study by Trp substitution and resulted in an increase and decrease in infectious virus titer, respectively, although these phenotypes were not the most dramatic in our panel. Interestingly, structure models give rise to incongruent hypotheses regarding the impact of G18W mutation (S1 Fig), suggesting this residue may provide another opportunity to further probe these structure models and test p7 function in cell culture and p7 ion channel activity after reconstitution in artificial membranes. Together our data indicate that escape mutants with significant fitness could be readily generated in the context of p7-targeting antiviral compounds, potentially limiting the efficacy of this class of inhibitors in the clinic. Still, there were several positions tested that did show a marked impact on infectious virus production; this was most pronounced when the residues within the first eighteen amino acids, comprising the N-terminal helical region, were interrogated.