New insights into the understanding of hepatitis C virus entry and cell-to-cell transmission by using the ionophore Monensin A.
Fiche publication
Date publication
juin 2015
Auteurs
Membres identifiés du Cancéropôle Est :
Pr BAUMERT Thomas
Tous les auteurs :
Feneant L, Potel J, Francois C, Sane F, Douam F, Belouzard S, Calland N, Vausselin T, Rouille Y, Descamps V, Baumert TF, Duverlie G, Lavillette D, Hober D, Dubuisson J, Wychowski C, Cocquerel L
Lien Pubmed
Résumé
In our study, we characterized the effect on the Hepatitis C Virus (HCV) life cycle of Monensin, an ionophore that is known to raise the intracellular pH. We show that Monensin inhibits HCV entry in a pangenotypic and dose-dependent manner. Monensin induces an alkalization of intracellular organelles, leading to an inhibition of the fusion step between viral and cellular membranes. Interestingly, we demonstrated that HCV cell-to-cell transmission is dependent on the vesicular pH. Under the selective pressure of Monensin, we selected a Monensin-resistant virus which has evolved to use a new entry route that is partially pH- and clathrin-independent. Characterization of this mutant led to the identification of two mutations in envelope proteins, Y297H in E1 and I399T in the hypervariable region 1 (HVR1) of E2, which confer resistance to Monensin and thus allow HCV to use a pH-independent entry route. Interestingly, the I399T mutation introduces a N-glycosylation site within HVR1, increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (ApoE) antibodies, suggesting that this mutation likely induces conformational changes in HVR1 that in turn modulate the association with ApoE. Strikingly, the I399T mutation dramatically reduces HCV cell-to-cell spread. In conclusion, we identified a mutation in HVR1 that overcomes the vesicular pH-dependence, modifies the biophysical properties of particles and drastically reduces cell-to-cell transmission, indicating that the regulation by HVR1 of particle association with ApoE might control the pH-dependence of cell-free and cell-to-cell transmission. Thus, HVR1 and ApoE are critical regulators of HCV propagation. IMPORTANCE: Although several cell surface proteins have been identified as entry factors for Hepatitis C Virus (HCV), precise mechanisms regulating its transmission into hepatic cells are still unclear. In our study, we used Monensin A, an ionophore that is known to raise the intracellular pH, and demonstrated that cell-free and cell-to-cell transmission are both pH-dependent processes. We generated Monensin-resistant viruses that displayed different entry routes and biophysical properties. Thanks to these mutants, we highlighted the importance of the hypervariable region 1 (HVR1) in E2 envelope protein for the association of particles with apolipoprotein E, which in turn might control the pH-dependency of cell-free and cell-to-cell transmission.
Référence
J Virol. 2015 Jun 3. pii: JVI.00192-15.