Towner Jonathan S, Brown David M, Nguyen Joseph H C, Semler Bert L
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697, USA.
Virology. 2003 Sep 15;314(1):432-42. doi: 10.1016/s0042-6822(03)00448-3.
In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required.
在本研究中，我们将1型脊髓灰质炎病毒（PV1）膜相关多肽3AB内的疏水结构域部分替换为人鼻病毒14型（HRV14）的类似序列。序列交换基于之前的一份报告，其中22个氨基酸的疏水区域被细分为两个结构域，I和II，后者被证明是膜结合所必需的（《生物化学杂志》271 (1996), 26810）。利用这些划分，将HRV14序列克隆到完整的1型脊髓灰质炎病毒cDNA序列中。从这些cDNA转录的RNA被转染到HeLa细胞单层中，并用于HeLa无细胞翻译/复制试验。数据表明，PV1和HRV14的3AB序列是可互换的；然而，这些替换会导致一系列显著的RNA复制缺陷，在某些情况下，还会导致蛋白质加工缺陷。将编码结构域替换的RNA转染到HeLa细胞单层后，收获病毒分离株，并对相应的病毒RNA进行测序。序列数据显示，对于羧基末端结构域替换（结构域II），在不同密码子的第一、第二和第三位鉴定出多个核苷酸变化。此外，数据表明，对于其中一种PV1/HRV14嵌合体的复制，可能需要脊髓灰质炎病毒蛋白2B内的补偿性突变。
