Mutations in hepatitis C virus p7 reduce both the egress and infectivity of assembled particles via impaired proton channel function

Abstract

Hepatitis C virus (HCV) p7 protein is critical for the efficient production of infectious virions in culture. p7 undergoes genotype-specific protein-protein interactions as well as displaying channel-forming activity, making it unclear whether the phenotypes of deleterious p7 mutations result from the disruption of one or both of these functions. Here, we showed that proton channel activity alone, provided in trans by either influenza virus M2 or genotype 1b HCV p7, was both necessary and sufficient to restore infectious particle production to genotype 2a HCV (JFH-1 isolate) carrying deleterious p7 alanine substitutions within the p7 dibasic loop (R33A, R35A), and the N-terminal trans-membrane region (N15 : C16 : H17/AAA). Both mutations markedly reduced mature p7 abundance, with those in the dibasic loop also significantly reducing levels of mature E2 and NS2. Interestingly, whilst M2 and genotype 1b p7 restored the same level of intracellular infectivity as JFH-1 p7, supplementing with the isogenic protein led to a further increase in secreted infectivity, suggesting a late-acting role for genotype-specific p7 protein interactions. Finally, cells infected by viruses carrying p7 mutations contained non-infectious corecontaining particles with densities equivalent to WT HCV, indicating a requirement for p7 proton channel activity in conferring an infectious phenotype to virions.

Cooperation between the Hepatitis C Virus p7 and NS5B Proteins Enhances Virion Infectivity

“…Both of these residues are located in the N-terminal transmembrane helix of p7 directly adjacent to a flexible hinge that connects this helix to transmembrane helix 2 in the protein (48). Further, these residues line the inner pore of the proton channel formed by these helices, and they are required for proton channel activity (47,48). We found that the C766Y change in p7 increased the overall size of lipid droplets in the infected cell.…”

“…However, the viral p7 transmembrane protein plays an important role in this process, as it is required for efficient virion assembly, release, and envelopment (27,41,45,46). The 63-amino-acid p7 protein oligomerizes to form a proton channel, and mutational analysis of p7 has revealed that this proton channel contributes to virion infectivity at a postassembly step (47). Interestingly, amino acid changes within p7 have been identified in many of the previous studies on culture adaptation of JFH-1 as contributing to increased viral titers (19,22,26,27,36).…”

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway

“…A mutation in the γ 1 34.5 viral protein of herpes simplex virus 1 was found to interfere with viral egress, causing majority of the viral particles to accumulate inside the cytoplasm58. Similarly, mutations in the hepatitis C viral protein p7 were found to impair proton channel function which resulted in decreased viral egress59. While we have identified a number of differences in the viral genome of MS and S41 strains (data not shown), their contribution in viral egress efficacy needs to be further investigated.…”

The elusive function of the hepatitis C virus p7 protein

“…Subsequent to core protein recruitment to LDs, core oligomerizes to form capsid structures; this presumably facilitates the formation of multi-order forms during the morphogenesis cycle that would include monomeric core, partial oligomers, fully oligomerized capsids and virions associated with triglycerides and β-lipoproteins, all of which would show different densities upon intracellular lysate fractionation (Andre et al, 2002;Gastaminza et al, 2006;Jones et al, 2011). We and others have shown that p7 mutations in TM1 and the cytoplasmic loop did not prevent the assembly of core-containing intracellular particles after iodixanol equilibrium gradient sedimentation (Atoom et al, 2013; Bentham et al, 2013). Compellingly, another report performed core quantitation on the fractions obtained after sucrosebased rate zonal fractionation and observed an increased proportion of incompletely assembled capsids.…”