Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation
Characterization of Determinants Important for Hepatitis C Virus p7 Function in Morphogenesis by Using trans-Complementation
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DOI：
10.1128/jvi.00691-09
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发表时间：
2009-11-15
影响因子：
5.4
通讯作者：
Pietschmann, Thomas
中科院分区：
文献类型：
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作者：
Brohm, Christiane;Steinmann, Eike;Pietschmann, Thomas
丙型肝炎病毒p7是一种完整的膜蛋白，在体外形成离子通道，对感染性病毒粒子的有效组装和释放至关重要。由于这些性质，p7被包括在病毒孔蛋白家族中，该家族包括甲型流感病毒M2和人类免疫缺陷病毒1型(HIV-1)VPU等蛋白质，这些蛋白质改变膜通透性并促进传染性病毒的释放。来自不同丙型肝炎病毒分离株的P7以不同的效率维持病毒的产生。此外，p7决定簇调控了E2/p7和p7/NS2信号肽切割位点的加工，并且E2/p7的切割是不完整的。因此，尚不清楚维持病毒产生的不同能力是由于不同的离子通道活动还是由于这些部位的交替处理。因此，我们开发了一种反式互补试验，允许在丙型肝炎病毒多蛋白之外分析p7，从而独立于处理。挽救p7缺陷的丙型肝炎病毒基因组是通过提供E2、p7和NS2，或者在某些情况下，在瞬时互补试验中以及在稳定的细胞系中仅通过p7来完成的。相反，甲型流感病毒M2和HIV-1 VPU都不能弥补丙型肝炎病毒形态发生中p7的缺陷。因此，p7对于产生具有感染性的丙型肝炎病毒颗粒是绝对必要的。此外，我们的数据表明，p7可以独立于上游信号序列工作，并且接近保守的二碱性基序p7的酪氨酸残基对于2a型病毒的最佳病毒生产是重要的。这里描述的实验系统应该有助于进一步研究p7的关键决定因素，这些决定因素对于p7的结构和功能是必不可少的，而不会因为改变的多蛋白加工而引起二次影响。
Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans-complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.