Fénéant Lucie, Potel Julie, François Catherine, Sané Famara, Douam Florian, Belouzard Sandrine, Calland Noémie, Vausselin Thibaut, Rouillé Yves, Descamps Véronique, Baumert Thomas F, Duverlie Gilles, Lavillette Dimitri, Hober Didier, Dubuisson Jean, Wychowski Czeslaw, Cocquerel Laurence
Molecular and Cellular Virology Laboratory, Center for Infection and Immunity of Lille, University Lille Nord de France, CNRS UMR8204, INSERM U1019, Pasteur Institute of Lille, Lille, France.
Virology Department, EA4294 UPJV, Amiens University Hospital, Amiens, France.
J Virol. 2015 Aug;89(16):8346-64. doi: 10.1128/JVI.00192-15. Epub 2015 Jun 3.
In our study, we characterized the effect of monensin, an ionophore that is known to raise the intracellular pH, on the hepatitis C virus (HCV) life cycle. We showed 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. Using 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, the Y297H mutation in E1 and the I399T mutation in 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 an N-glycosylation site within HVR1 and increases the density of virions and their sensitivity to neutralization with anti-apolipoprotein E (anti-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 summary, 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.
Although several cell surface proteins have been identified as entry factors for hepatitis C virus (HCV), the precise mechanisms regulating its transmission to 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 pathways 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 hypervariable region 1 (HVR1) of the 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.
在我们的研究中，我们描述了莫能菌素（一种已知可提高细胞内pH值的离子载体）对丙型肝炎病毒（HCV）生命周期的影响。我们发现莫能菌素以泛基因型和剂量依赖性方式抑制HCV进入。莫能菌素诱导细胞内细胞器碱化，导致病毒膜与细胞膜之间融合步骤受到抑制。有趣的是，我们证明HCV细胞间传播依赖于囊泡pH值。利用莫能菌素的选择压力，我们筛选出一种对莫能菌素耐药的病毒，该病毒已进化出使用一种部分不依赖pH值和网格蛋白的新进入途径。对该突变体的特征分析导致在包膜蛋白中鉴定出两个突变，E1中的Y297H突变和E2高变区1（HVR1）中的I399T突变，这赋予了对莫能菌素的抗性，从而使HCV能够使用不依赖pH值的进入途径。有趣的是，I399T突变在HVR1内引入了一个N-糖基化位点，并增加了病毒粒子的密度及其对抗载脂蛋白E（抗ApoE）抗体中和的敏感性，这表明该突变可能诱导HVR1中的构象变化，进而调节与ApoE的结合。引人注目的是，I399T突变显著降低了HCV细胞间传播。总之，我们在HVR1中鉴定出一个突变，该突变克服了囊泡pH值依赖性，改变了病毒粒子的生物物理特性，并大幅降低了细胞间传播，表明HVR1对病毒粒子与ApoE结合的调节可能控制游离病毒和细胞间传播的pH值依赖性。因此，HVR1和ApoE是HCV传播的关键调节因子。
尽管已鉴定出几种细胞表面蛋白作为丙型肝炎病毒（HCV）的进入因子，但其向肝细胞传播的精确调控机制仍不清楚。在我们的研究中，我们使用了莫能菌素A（一种已知可提高细胞内pH值的离子载体），并证明游离病毒和细胞间传播途径都是依赖pH值的过程。我们产生了具有不同进入途径和生物物理特性的莫能菌素耐药病毒。借助这些突变体，我们强调了E2包膜蛋白高变区1（HVR1）对于病毒粒子与载脂蛋白E结合的重要性，这反过来可能控制游离病毒和细胞间传播的pH值依赖性。
