School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, LS2 9JT, UK.
Present address: Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
J Gen Virol. 2023 Oct;104(10). doi: 10.1099/jgv.0.001909.
Chikungunya virus (CHIKV) is an alphavirus, transmitted by species mosquitoes. The CHIKV single-stranded positive-sense RNA genome contains two open reading frames, coding for the non-structural (nsP) and structural proteins of the virus. The non-structural polyprotein precursor is proteolytically cleaved to generate nsP1-4. Intriguingly, most isolates of CHIKV (and other alphaviruses) possess an opal stop codon close to the 3' end of the nsP3 coding sequence and translational readthrough is necessary to produce full-length nsP3 and the nsP4 RNA polymerase. Here we investigate the role of this stop codon by replacing the arginine codon with each of the three stop codons in the context of both a subgenomic replicon and infectious CHIKV. Both opal and amber stop codons were tolerated in mammalian cells, but the ochre was not. In mosquito cells all three stop codons were tolerated. Using SHAPE analysis we interrogated the structure of a putative stem loop 3' of the stop codon and used mutagenesis to probe the importance of a short base-paired region at the base of this structure. Our data reveal that this stem is not required for stop codon translational readthrough, and we conclude that other factors must facilitate this process to permit productive CHIKV replication.
基孔肯雅病毒（CHIKV）是一种甲病毒，由 种蚊子传播。CHIKV 的单链正链 RNA 基因组包含两个开放阅读框，编码病毒的非结构（nsP）和结构蛋白。非结构多蛋白前体被蛋白水解切割，生成 nsP1-4。有趣的是，大多数 CHIKV（和其他甲病毒）分离株在 nsP3 编码序列的 3' 端附近具有一个琥珀终止密码子，并且需要翻译通读才能产生全长 nsP3 和 nsP4 RNA 聚合酶。在这里，我们通过在亚基因组复制子和传染性 CHIKV 的背景下用每个终止密码子替换精氨酸密码子来研究这个终止密码子的作用。在哺乳动物细胞中，琥珀和琥珀终止密码子都被容忍，但赭石则不能。在蚊子细胞中，所有三种终止密码子都被容忍。我们使用 SHAPE 分析来研究终止密码子 3' 处一个假定茎环结构的结构，并使用诱变来探测该结构底部一个短碱基配对区域的重要性。我们的数据表明，这个茎对于终止密码子的翻译通读不是必需的，我们得出结论，其他因素必须促进这个过程，以允许有效的 CHIKV 复制。
