Abstract
Viroporins are small transmembrane proteins with ion channel activities modulating properties of intracellular membranes that have diverse proviral functions. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles (VP). HCV p7 is released from the viral polyprotein through cleavage at E2-p7 and p7-NS2 junctions by signal peptidase, but also exists as an E2p7 precursor, of poorly defined properties. Here, we found that ectopic p7 expression in HCVcc-infected cells reduced secretion of particle-associated E2 glycoproteins. Using biochemical assays, we show that p7 dose-dependently slows down the ER-to-Golgi traffic, leading to intracellular retention of E2, which suggested that timely E2p7 cleavage and p7 liberation are critical events to control E2 levels. By studying HCV mutants with accelerated E2p7 processing, we demonstrate that E2p7 cleavage controls E2 intracellular expression and secretion levels of nucleocapsid-free subviral particles and infectious virions. In addition, our imaging data reveal that, following p7 liberation, the amino-terminus of p7 is exposed towards the cytosol and coordinates the encounter between NS5A and NS2-based assembly sites loaded with E1E2 glycoproteins, which subsequently leads to nucleocapsid envelopment. We identify punctual mutants at p7 membrane interface that, by abrogating NS2/NS5A interaction, are defective for transmission of infectivity owing to decreased secretion of core and RNA and to increased secretion of non/partially-enveloped particles. Altogether, our results indicate that the retarded E2p7 precursor cleavage is essential to regulate the intracellular and secreted levels of E2 through p7-mediated modulation of the cell secretory pathway and to unmask critical novel assembly functions located at p7 amino-terminus.
Highlights
Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases worldwide
We have explored how the retarded cleavage between E2 glycoprotein and p7 viroporin could regulate their functions associated to virion assembly and/or perturbation of cellular membrane processes
Our study identifies a novel assembly function located at p7 amino-terminus that is unmasked through E2p7-regulated processing and that controls the infectivity of different types of released viral particles
Summary
Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases worldwide. At least three precursors, i.e., tandem proteins with delayed cleavage, are detected and may implement functions different than their cognate individual proteins. They consist of immature core protein, associated to the D3 trans-membrane peptide, whose removal allows core targeting to lipid droplets (LDs) [4, 5]; NS4B-5A, which promotes the formation of replication vesicles [6]; and E2p7 [7,8,9,10,11], whose properties are explored in this report
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