Entry and replication of positive-sense, RNA viruses

丙型肝炎病毒是一个重大的全球公共卫生问题，全世界约有7000万人感染。目前还没有批准的疫苗来对抗丙型肝炎病毒感染。疾病控制中心估计，仅在美国每年就有超过4万名新感染者，世界其他地区还有150万名新感染者。慢性丙型肝炎病毒感染是可以通过有效的、尽管昂贵的抗病毒治疗来治愈的。然而，药物治疗并不是在世界范围内根除丙型肝炎病毒感染的可行途径，因为这样做的成本将令人望而却步。此外，成功治疗感染了一种病毒的患者并不排除再次感染另一种病毒。药物治疗方法也因以下事实而变得复杂：大多数受影响的人没有意识到自己被感染了，许多人从事危险行为，如静脉吸毒。简而言之，最好的长期解决方案是投入大量的智力和财政资源，发现和开发一种对大多数(如果不是全部)丙型肝炎病毒基因有效的多价疫苗。 丙型肝炎病毒通过一个多步骤进入肝细胞，需要一系列宿主细胞因子和病毒包膜糖蛋白E1和E2。丙型肝炎病毒糖蛋白介导细胞靶向、内吞作用和最终由内体酸化刺激的膜融合。至少有四种细胞因子对丙型肝炎病毒的附着和进入至关重要：CD81、B型清道夫受体I(SRBI)、claudin-1(CLDN)和occludin(OCLN)。CD81在多种细胞系中普遍表达，表明CD81的作用仅次于肝细胞特异性受体结合。CD81与病毒粒子一起转移到紧密连接处，并与内体中的迟进入因子CLDN和OCLN接触，以进行酸化和进入。大多数广谱中和抗体排除了丙型肝炎病毒E2与细胞受体CD81的大细胞外环(LEL)之间的相互作用。我们观察到低pH增强了CD81-LEL与E2的结合，并确定了E2/CD81-LEL的晶体结构。当与CD81结合时，E2沿着CD81表面延伸一个内环。将E2/CD81-LEL复合体对接到嵌入的全长CD81膜上，将环放置在膜的近端。脂质体浮选实验表明，低pH和CD81-LEL增强了E2与膜的相互作用，而高度保守的疏水氨基酸的结构突变体则取消了膜结合。这些数据支持一种模型，即酸化和受体结合导致E2在膜融合准备过程中的构象变化。此后，介导丙型肝炎病毒细胞进入和膜融合的分子机制仍不清楚。这些综合的结构、生化和生物物理结果为更好地确定包膜糖蛋白在丙型肝炎病毒感染中的功能作用为疫苗设计奠定了基础。

HCV represents a major global public health problem, infecting approximately 70 million people worldwide. There is currently no approved vaccine to counter HCV infection. The Center for Disease Control estimates more than 40,000 new infections annually in the US alone with an additional 1.5 million new infections in the rest of world. Chronic HCV infection is curable by an effective, albeit expensive, antiviral therapy. Drug treatment is not, however, a feasible route to worldwide eradication of HCV infection, as the cost of doing so would be prohibitive. Moreover, successful treatment of a patient infected with one viral genotype does not preclude re-infection with another. The drug treatment approach is also complicated by the fact that most affected individuals are unaware that they are infected, and many engage in risky behaviors, such as intravenous drug use. Simply put, the best long term solution is to invest considerable intellectual and financial resources in discovery and development of a polyvalent vaccine effective against most, if not all, HCV viral genotypes. HCV enters hepatocytes through a multistep process requiring a series of host cellular factors and the viral envelope glycoproteins E1 and E2. The HCV glycoproteins mediate cell targeting, endocytosis, and membrane fusion ultimately stimulated by endosomal acidification. At least four cellular factors are critical for HCV attachment and entry: CD81, scavenger receptor class B type I (SRBI), claudin-1 (CLDN), and occludin (OCLN). CD81 is ubiquitously expressed on a variety of cell lines, indicating a role secondary to hepatocyte-specific receptor binding. CD81 translocates with the virion to tight junctions and engages with late entry factors, CLDN and OCLN, in the endosome for acidification and entry. The majority of broadly neutralizing antibodies preclude interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81. We observed that low pH enhances CD81-LEL binding to E2 and determined the crystal structure of E2/CD81-LEL. Upon binding CD81, E2 extends an internal loop along the surface of CD81. Docking of the E2/CD81-LEL complex onto a membrane embedded, full length CD81 places the loop proximal to the membrane. Liposome flotation assays demonstrate that low pH and CD81-LEL increase E2 interaction with membranes, while structure-based mutants of highly conserved, hydrophobic amino acids abolish membrane binding. These data support a model that acidification and receptor binding result in a conformation change in E2 in preparation for membrane fusion.The molecular mechanisms thereafter, for mediating cell entry and membrane fusion for HCV, remain undefined. These comprehensive structural, biochemical, and biophysical results have established a foundation to better define the functional roles of the envelope glycoproteins in HCV infection for vaccine design.