Department of Biology, York Universitygrid.21100.32, Toronto, Ontario, Canada.
J Virol. 2021 Sep 27;95(20):e0103421. doi: 10.1128/JVI.01034-21. Epub 2021 Aug 4.
One of the many challenges faced by RNA viruses is the maintenance of their genomes during infections of host cells. Members of the family are plus-strand RNA viruses with unmodified triphosphorylated genomic 5' termini. The tombusvirus was used to investigate how it protects its RNA genome from attack by 5'-end-targeting degradation enzymes. and assays were employed to determine the role of genomic RNA structure in conferring protection from the 5'-to-3' exoribonuclease Xrn. The results revealed that (i) the CIRV RNA genome is more resistant to Xrn than its sg mRNAs, (ii) the genomic 5'-untranslated region (UTR) folds into a compact RNA structure that effectively and independently prevents Xrn access, (iii) the RNA structure limiting 5' access is formed by secondary and tertiary interactions that function cooperatively, (iv) the structure is also able to block access of RNA pyrophosphohydrolase to the genomic 5' terminus, and (v) the RNA structure does not stall an actively digesting Xrn. Based on its proficiency at impeding Xrn 5' access, we have termed this 5'-terminal structure an rn-vading RNA, or xeRNA. These and other findings demonstrate that the 5'UTR of the CIRV RNA genome folds into a complex structural conformation that helps to protect its unmodified 5' terminus from enzymatic decay during infections. The plus-strand RNA genomes of plant viruses in the large family are not 5' capped. Here, we explored how a species in the type genus protects its genomic 5' end from cellular nuclease attack. Our results revealed that the 5'-terminal sequence of the CIRV genome folds into a complex RNA structure that limits access of the 5'-to-3' exoribonuclease Xrn, thereby protecting it from processive degradation. The RNA conformation also impeded access of RNA pyrophosphohydrolase, which converts 5'-triphosphorylated RNA termini into 5'-monophosphorylated forms, the preferred substrate for Xrn. This study represents the first report of a higher-order RNA structure in an RNA plant virus genome independently conferring resistance to 5'-end-attacking cellular enzymes.
RNA 病毒面临的众多挑战之一是在感染宿主细胞时维持其基因组。家族成员是具有未经修饰的三磷酸化基因组 5' 末端的正链 RNA 病毒。利用番茄丛矮病毒来研究其如何保护其 RNA 基因组免受 5'-端靶向降解酶的攻击。使用 和 测定来确定基因组 RNA 结构在赋予对 5'-3' 外切核酸酶 Xrn 的抗性方面的作用。结果表明：(i) CIRV RNA 基因组比其 sg mRNAs 更能抵抗 Xrn，(ii) 基因组 5'-非翻译区 (UTR) 折叠成一种紧凑的 RNA 结构，可有效且独立地阻止 Xrn 进入，(iii) 限制 5' 进入的 RNA 结构由二级和三级相互作用形成，协同作用，(iv) 该结构还能够阻止 RNA 焦磷酸水解酶进入基因组 5' 末端，以及 (v) RNA 结构不会阻止正在积极消化的 Xrn。基于其阻止 Xrn 5' 进入的能力，我们将这种 5' 末端结构称为 rn-vading RNA，或 xeRNA。这些和其他发现表明，CIRV RNA 基因组的 5'UTR 折叠成一种复杂的结构构象，有助于保护其未经修饰的 5' 末端在感染过程中免受酶促降解。在大型家族中的植物病毒的正链 RNA 基因组没有 5' 帽。在这里，我们探讨了属中的一个种如何保护其基因组 5' 端免受细胞核酸酶的攻击。我们的结果表明，CIRV 基因组的 5' 末端序列折叠成一种复杂的 RNA 结构，限制了 5'-3' 外切核酸酶 Xrn 的进入，从而保护其免受连续降解。该 RNA 构象还阻碍了 RNA 焦磷酸水解酶的进入，该酶将 5'-三磷酸化 RNA 末端转化为 5'-单磷酸化形式，这是 Xrn 的首选底物。这项研究代表了第一个独立赋予 RNA 植物病毒基因组抵抗 5'-端攻击细胞酶的高级别 RNA 结构的报告。
