The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences
The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences
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DOI：
10.1073/pnas.1834545100
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发表时间：
2003-09-30
影响因子：
11.1
通讯作者：
Bukh, J
中科院分区：
文献类型：
--
作者：
Sakai, A;St Claire, MS;Bukh, J
丙型肝炎病毒（HCV）p7蛋白在病毒生命周期中的作用尚不清楚。先前的体外数据表明，这63个氨基酸的多肽位于内质网中，并有两个跨膜结构域（TMD）连接的细胞质环，氨基和羧基末端的尾巴是面向内质网腔。此外，最近的体外研究表明，HCV p7可以作为病毒编码的离子通道。因此，它可能是未来药物开发的相关目标。我们研究了HCV p7在体内的作用。由于HCV在细胞培养中不能有效复制，我们诱变了感染性基因型la cDNA克隆的p7，并通过肝内转染测试了每个突变体在黑猩猩中的感染性的RNA转录本。通过对转染的哺乳动物细胞的研究证实了突变多肽的适当加工。缺失全部或部分p7的突变体和细胞质环中两个保守残基取代的突变体不可行。因此，p7对于HCV的感染性是必需的。其中Ia克隆的p7被来自感染性基因型2a克隆的p7替换的嵌合体也是不能存活的。这一发现表明p7和其他基因组区域之间存在基因型特异性相互作用。为了确定p7的哪些部分在这种相互作用中发挥了最重要的作用，我们测试了三种具有1a骨架的嵌合体，其中只有p7的特定结构域具有2a序列。具有2a尾和TMD以及1a胞质环的p7嵌合体是不能存活的。具有2a尾和胞质环以及la TMD的突变体也不能存活。然而，具有2a TMD和胞质环和la尾的p7嵌合体是存活的。转染的黑猩猩在第2周出现病毒血症，并且恢复的病毒具有嵌合序列。这些数据表明，氨基和/或羧基末端的管腔内尾巴的p7含有基因型特异性功能的序列。
The role of the hepatitis C virus (HCV) p7 protein in the virus life cycle is not known. Previous in vitro data indicated that this 63-aa polypeptide is located in the endoplasmic reticulum and has two transmembrane domains (TMDs) connected by a cytoplasmic loop; the amino- and carboxyl-terminal tails are oriented toward the endoplasmic reticulum lumen. Furthermore, recent in vitro studies suggested that HCV p7 could function as a virus-encoded ion channel. It might therefore be a relevant target for future drug development. We studied the role of HCV p7 in vivo. Because HCV does not replicate efficiently in cell culture, we mutagenized p7 of an infectious genotype la cDNA clone and tested RNA transcripts of each mutant for infectivity in chimpanzees by intrahepatic transfection. Appropriate processing of mutant polypeptides was confirmed by studies in transfected mammalian cells. Mutants with deletions of all or part of p7 and a mutant with substitutions of two conserved residues in the cytoplasmic loop were not viable. Thus, p7 is essential for infectivity of HCV. A chimera in which the p7 of the 1 a clone was replaced with p7 from an infectious genotype 2a clone also was not viable. This finding suggests a genotype-specific interaction between p7 and other genomic regions. To define which portions of p7 played the most significant role for this interaction, we tested three chimeras with the 1 a backbone in which only specific domains of p7 had the 2a sequence. A p7 chimera with 2a tails and TMDs and the 1a cytoplasmic loop was not viable. A mutant with 2a tails and cytoplasmic loop and la TMDs also was not viable. However, a p7 chimera with 2a TMDs and cytoplasmic loop and la tails was viable. The transfected chimpanzee became viremic at week 2, and recovered viruses had the chimeric sequence. These data indicate that the amino- and/or carboxyl-terminal intraluminal tails of p7 contain sequences with genotype-specific function.