Paudel Dinesh Babu, Sanfaçon Hélène
Department of Botany, University of British Columbia, Vancouver, BC, Canada.
Summerland Research and Development Centre, Agriculture and Agri-Food Canada, Summerland, BC, Canada.
PLoS One. 2021 Apr 9;16(4):e0249928. doi: 10.1371/journal.pone.0249928. eCollection 2021.
Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5'-viral genome-linked protein (VPg) and have a 3' polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3' untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5' regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5' region and 3' UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5' region and 3' UTR of RNA2 are required for efficient cap-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion virus, another nepovirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two viruses.
番茄环斑病毒（ToRSV，属于线虫传多面体病毒属，矮化病毒科，小RNA病毒目）是一种二分体正链RNA病毒，每个RNA编码一个大的多聚蛋白。ToRSV RNA与一个5'-病毒基因组连接蛋白（VPg）相连，并具有3' polyA尾，提示一种非典型的不依赖帽子结构的翻译起始机制。RNA1和RNA2的3'非翻译区（UTR）异常长（约1.5 kb），并共享几段大的序列同源性。在病毒RNA的5'区域存在几个推定的符合读框的起始密码子，这在两个RNA之间也高度保守。利用含有ToRSV Rasp1分离株（ToRSV-Rasp1）RNA2的5'区域和3' UTR的报告转录本以及体外小麦胚提取物翻译试验，我们提供了证据表明，尽管密码子上下文不佳，但翻译仅在第一个AUG起始。我们还表明，RNA2的5'区域和3' UTR对于这些转录本的高效不依赖帽子结构的翻译都是必需的。我们在RNA2多聚蛋白的5'近端编码区和RNA2 3' UTR中鉴定出翻译增强元件。当由RNA2 3' UTR组成的RNA以反式提供时，对照报告转录本的依赖帽子结构的翻译受到抑制。综合来看，我们的结果表明在ToRSV-Rasp1 RNA2 3' UTR中存在一个顺式作用元件（CITE），它招募一个或几个翻译因子，并与RNA的5'区域一起促进高效的不依赖帽子结构的翻译。非重叠缺失诱变将推定的CITE定位到1547 nt长的3' UTR的一个200 nt片段（第773-972位核苷酸）。我们得出结论，ToRSV RNA2翻译起始的一般机制与先前报道的另一种线虫传多面体病毒——黑醋栗退化病毒的RNA的机制相似。然而，两种病毒之间翻译增强元件的位置、序列和预测结构有所不同。
