Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion
Hepatitis C Virus Envelope Glycoprotein E1 Forms Trimers at the Surface of the Virion
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DOI：
10.1128/jvi.00991-15
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发表时间：
2015-10-01
影响因子：
5.4
通讯作者：
Penin, Francois
中科院分区：
文献类型：
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作者：
Falson, Pierre;Bartosch, Birke;Penin, Francois
在丙型肝炎病毒感染的细胞中，包膜糖蛋白E1和E2以异源二聚体的形式组装。为了研究病毒粒子相关包膜蛋白寡聚的潜在变化，我们在还原条件下进行了SDS-PAGE，但没有热变性。这揭示了在细胞培养的丙型肝炎病毒(HCVcc)以及在丙型肝炎病毒假颗粒(HCVpp)的背景下，存在对十二烷基硫酸钠耐药的E1三聚体。E1三聚体的形成依赖于E2三聚体的共表达。为了进一步了解E1三聚体形成的起源，我们在细菌中表达了与报告蛋白融合的E1(TME1)和E2(TME2)的跨膜(TM)结构域，并用SDS-PAGE和Western blotting对融合蛋白进行了分析。正如预期的那样，对于强相互作用的TM结构域，观察到了对SDS具有抗性的TME1-TME2杂二聚体。这些分析还揭示了TME1的同源二聚体和同源三聚体，表明这种络合物是稳定的物种。TME1的N-末端片段显示一个高度保守的GxxxG序列，该基序被认为参与了膜内蛋白质-蛋白质的相互作用。该基序中甘氨酸残基(Gly354和Gly358)的单或双突变显著减少或取消细菌中TME1同源三聚体的形成，以及HCVpp和HCVcc系统中E1同源三聚体的形成。随之而来的是感染性的丧失，表明E1三聚体对于病毒的感染性是必不可少的。综上所述，这些结果表明，E1E2异源二聚体在丙型肝炎病毒颗粒上形成三聚体，支持了E1可能是一种融合蛋白的假说。在受感染的细胞中，这两种蛋白形成了一个复合体，其中E2与细胞受体相互作用，而E1的功能仍然知之甚少。然而，最近的结构数据表明，E1可能是负责病毒和细胞膜之间融合过程的蛋白质。在这里，我们研究了丙型肝炎病毒包膜糖蛋白的低聚状态。我们证明了在病毒粒子组装后，E1形成了功能三聚体，除了对E2的要求外，这种寡聚的决定因素存在于位于E1跨膜区的保守的GxxxG基序中。综上所述，这些结果表明，在丙型肝炎病毒颗粒组装过程中，可能会发生E1E2异源二聚体复合体的重排，从而产生E1E2异源二聚体的三聚体。获得关于这个三聚体的结构信息将有助于抗丙型肝炎病毒疫苗的设计。
In hepatitisCvirus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV(HCVcc) as well as in the context of HCV pseudo particles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1-TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein.IMPORTANCEHCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.