Denolly, Solène, Mialon, Chloé, Bourlet, Thomas, Amirache, Fouzia, Penin, François, Lindenbach, Brett, Boson, Bertrand, Cosset, François-Loïc, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP), Université Jean Monnet [Saint-Étienne] (UJM), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Department of Microbial Pathogenesis, Yale University School of Medicine, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Jean Monnet - Saint-Étienne (UJM), and Yale School of Medicine [New Haven, Connecticut] (YSM)
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., Author summary Viroporins are small transmembrane viral proteins with ion channel activities modulating properties of intracellular membranes, which impacts several fundamental biological processes such as trafficking, ion fluxes as well as connections and exchanges between organelles. Hepatitis C virus (HCV) encodes a viroporin, p7, acting during assembly, envelopment and secretion of viral particles. HCV p7 is produced by cleavage from the HCV polyprotein but also exists as an E2p7 precursor, of poorly defined properties. In this study, 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. Specifically, we demonstrate that p7 is able to regulate the cell secretory pathway, which induces the intracellular retention of HCV glycoproteins and favors assembly of HCV particles. Our study also 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. Altogether, our results underscore a critical post-translational control of assembly and secretion of HCV particles that governs their specific infectivity.