Hepatitis C Virus p7 Protein Is Crucial for Assembly and Release of Infectious Virions

Abstract

Hepatitis C virus (HCV) infection is associated with chronic liver disease and currently affects about 3% of the world population. Although much has been learned about the function of individual viral proteins, the role of the HCV p7 protein in virus replication is not known. Recent data, however, suggest that it forms ion channels that may be targeted by antiviral compounds. Moreover, this protein was shown to be essential for infectivity in chimpanzee. Employing the novel HCV infection system and using a genetic approach to investigate the function of p7 in the viral replication cycle, we find that this protein is essential for efficient assembly and release of infectious virions across divergent virus strains. We show that p7 promotes virus particle production in a genotype-specific manner most likely due to interactions with other viral factors. Virus entry, on the other hand, is largely independent of p7, as the specific infectivity of released virions with a defect in p7 was not affected. Together, these observations indicate that p7 is primarily involved in the late phase of the HCV replication cycle. Finally, we note that p7 variants from different isolates deviate substantially in their capacity to promote virus production, suggesting that p7 is an important virulence factor that may modulate fitness and in turn virus persistence and pathogenesis.

Viroporins: structure, function and potential as antiviral targets

“…The advent of HCV infectious culture based on the genotype 2a 'JFH-1' (Japanese fulminant hepatitis) infectious isolate (Wakita et al, 2005) led to the identification of an essential role for p7 during the production of infectious HCV particles (Jones et al, 2007;Steinmann et al, 2007a). Viable full-length HCV containing IRES elements inserted between E2 and p7 or p7 and NS2 argued against a functional role for p7 precursors (Jones et al, 2007).…”

“…Viable full-length HCV containing IRES elements inserted between E2 and p7 or p7 and NS2 argued against a functional role for p7 precursors (Jones et al, 2007). Both early-and late-acting defects in virion production have been described where p7 was (partially) deleted, mutated at specific residues or treated with inhibitors (Bentham et al, 2013;Foster et al, 2011Foster et al, , 2014Jones et al, 2007;Steinmann et al, 2007a;Vieyres et al, 2013;Wozniak et al, 2010). This is now known to result from p7 performing multiple functions within infected cells, comprising distinct protein-protein interactions as well as its channel-forming activity.…”

“…p7 channel activity appears to influence a late-acting phase of the HCV life cycle, distinct from that concerning protein-protein interactions; whereas p7 deletions and deleterious point mutations abrogate infectivity in all compartments (Atoom et al, 2013;Bentham et al, 2013;Brohm et al, 2009;Jones et al, 2007;Steinmann et al, 2007a;Wozniak et al, 2010), small-molecule p7 inhibitors prevent the accumulation of secreted, but not intracellular infectivity (Foster et al, 2011(Foster et al, , 2014. Point mutations recapitulating the p7 inhibitor-induced phenotype have not been identified, yet unlike (partial) deletion mutants (Brohm et al, 2009), infectivity of HCV carrying mutations to the basic loop region (to either Ala or the less hydrophobic Glu) can be partially restored by trans-complementation with IAV M2 or by treating cells with the V-type ATPase inhibitor bafilomycin A (Bentham et al, 2013;Wozniak et al, 2010).…”

“…Whilst this clearly depends upon antibody detection limits, with potential interference from HCV glycoproteins, a similar outcome resulted from studies of the related pestivirus, bovine viral diarrhea virus (BVDV) (Elbers et al, 1996). Furthermore, high efficiency particle-producing chimaeric HCV strains yield measurable infectivity despite carrying p7 basic loop mutations (albeit with *1000-fold reduction in titre); mutant-derived virions possessed equivalent specific infectivity to that of WT chimaeric HCV (Steinmann et al, 2007a). However, loop mutations likely disrupt p7 channel activity indirectly rather than by the formation of inactive channel complexes, via effects upon protein processing/stability and membrane insertion (Bentham et al, 2013;Pérez-Berná et al, 2008;StGelais et al, 2009).…”

Hepatitis c virus and host cell lipids: An intimate connection

“…[31][32][33][34] The p7 protein, composed of two TM regions that are interconnected by a short cytoplasmic loop, 35 is a viroporin able to form hexa-and heptameric complexes serving as ion channels and required for virus assembly and release. 36,37 NS2 is a dimeric integral membrane protein, composed of two functionally and topologically distinct domains: a highly hydrophobic, N-terminal, membrane anchor domain and a C-terminal cysteine protease domain, which liberates the C-terminus of NS2 from NS3. 38,39 NS2 is dispensable for genome replication, 4 but plays a central role in assembly, for which its protease activity is not required.…”