Schlesinger M J, London S D, Ryan C
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Virology. 1993 Mar;193(1):424-32. doi: 10.1006/viro.1993.1139.
Encoded in the genomes of all alphaviruses is a hydrophobic polypeptide of 55 amino acids, which is post-translationally modified with 4 covalently bound palmitic acids. This protein, noted as 6K, associates with membranes and is transported along with the two virus transmembranal glycoproteins to the site of virus assembly at the infected cell's plasma membrane. Previous studies showed that mutations in the 6K protein led to the slow release of aberrant, multi-cored infectious virions. In this paper, we report that an in-frame insertion of 45 nucleotides into an internal site of the 6K gene of Sindbis virus produced single-cored infectious particles at about 5% the yield of wild-type virus when the mutant was grown on avian, mammalian, and insect cells. Although the 15 amino acids were inserted at position 29 of the 55-amino-acid 6K protein, the mutation interfered with the cotranslational proteolytic processing that cleaves the 6K at its amino terminus from the Sindbis virus p62 glycoprotein and at its carboxyl terminus from the E1 glycoprotein. As a result, the amounts of normal p62 and E1 proteins were only half that made in cells infected with wild-type virus. In addition, the post-translational proteolytic conversion of p62 to E2 occurred at 10% the rate of wild-type proteins and the extensive fatty acylation normally detected on wild-type 6K protein was not found on the altered 6K protein. None of the mutated 6K protein was detected in virions, which were morphologically indistinguishable from wild-type virus. The mutant 6K virions also were similar to wild type in their rate of attachment, uncoating, and formation of an early nonstructural virus protein in avian cells. When compared with the wild-type virus, 6K29-infected cells exhibited a decreased rate of host-cell protein synthesis shut off. However, the rates of virus capsid synthesis were the same, indicating that capsid protein, per se, is not involved in shut off of host-cell protein synthesis. In complementation studies, this mutant exhibited a trans-dominant phenotype. These data provide clues about the topology of 6K protein in the membrane and its function in virus maturation.
所有甲病毒的基因组中都编码有一种由55个氨基酸组成的疏水多肽，该多肽在翻译后会被4个共价结合的棕榈酸修饰。这种被称为6K的蛋白质与膜结合，并与两种病毒跨膜糖蛋白一起被转运到受感染细胞的质膜处的病毒组装位点。先前的研究表明，6K蛋白中的突变会导致异常的、多核感染性病毒粒子的缓慢释放。在本文中，我们报告称，当该突变体在禽类、哺乳动物和昆虫细胞中生长时，在辛德毕斯病毒6K基因的一个内部位点框内插入45个核苷酸，产生了单核感染性颗粒，其产量约为野生型病毒的5%。尽管在由55个氨基酸组成的6K蛋白的第29位插入了15个氨基酸，但该突变干扰了共翻译蛋白水解过程，该过程在6K的氨基末端将其从辛德毕斯病毒p62糖蛋白上切割下来，并在其羧基末端将其从E1糖蛋白上切割下来。结果，正常p62和E1蛋白的量仅为感染野生型病毒的细胞中产生量的一半。此外，p62向E2的翻译后蛋白水解转化速率仅为野生型蛋白的10%，并且在改变后的6K蛋白上未发现通常在野生型6K蛋白上检测到的广泛的脂肪酰化。在病毒粒子中未检测到任何突变的6K蛋白，其形态与野生型病毒无法区分。突变的6K病毒粒子在禽类细胞中的附着、脱壳以及早期非结构病毒蛋白形成的速率也与野生型相似。与野生型病毒相比，感染6K29的细胞表现出宿主细胞蛋白质合成关闭速率降低。然而，病毒衣壳合成速率相同，这表明衣壳蛋白本身不参与宿主细胞蛋白质合成的关闭。在互补研究中，该突变体表现出反式显性表型。这些数据为6K蛋白在膜中的拓扑结构及其在病毒成熟中的功能提供了线索。
