Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA.
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA
J Virol. 2021 Apr 12;95(9). doi: 10.1128/JVI.02109-20.
(OPMV) is a recently discovered umbravirus in the family OPMV has a plus-sense genomic RNA (gRNA) of 4,241 nucleotides (nt) from which replication protein p35 and p35 extension product p98, the RNA-dependent RNA polymerase (RdRp), are expressed. Movement proteins p27 (long distance) and p28 (cell to cell) are expressed from a 1,440-nt subgenomic RNA (sgRNA2). A highly conserved structure was identified just upstream from the sgRNA2 transcription start site in all umbraviruses, which includes a carmovirus consensus sequence, denoting generation by an RdRp-mediated mechanism. OPMV also has a second sgRNA of 1,554 nt (sgRNA1) that starts just downstream of a canonical exoribonuclease-resistant sequence (xrRNA). sgRNA1 codes for a 30-kDa protein that is in frame with p28 and cannot be synthesized in other umbraviruses. Eliminating sgRNA1 or truncating the p30 open reading frame (ORF) without affecting p28 substantially reduced accumulation of OPMV gRNA, suggesting a functional role for the protein. The 652-nt 3' untranslated region of OPMV contains two 3' cap-independent translation enhancers (3' CITEs), a T-shaped structure (TSS) near its 3' end, and a -like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences , which differs from how umbravirus 3' CITEs were used in a previous study. Similarly to most 3' CITEs, the OPMV BTE links to the 5' end via a long-distance RNA-RNA interaction. Analysis of 14 BTEs revealed additional conserved sequences and structural features beyond the previously identified 17-nt conserved sequence. (OPMV) is an umbravirus in the family We determined that OPMV accumulates two similarly sized subgenomic RNAs (sgRNAs), with the smaller known to code for proteins expressed from overlapping open reading frames. The slightly larger sgRNA1 has a 5' end just upstream from a previously predicted xrRNA site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNA sites, only sgRNA1 of OPMV can code for a protein that is an extension product of umbravirus ORF4. Inability to generate the sgRNA or translate this protein was associated with reduced gRNA accumulation We also characterized the OPMV BTE structure, a 3' cap-independent translation enhancer (3' CITE). Comparisons of 13 BTEs with the OPMV BTE revealed additional stretches of sequence similarity beyond the 17-nt signature sequence, as well as conserved structural features not previously recognized in these 3' CITEs.
（OPMV）是一种最近发现的 Umbravirus，其基因组 RNA（gRNA）为 4241 个核苷酸（nt），其中表达复制蛋白 p35 和 p35 延伸产物 p98、RNA 依赖性 RNA 聚合酶（RdRp）。移动蛋白 p27（远距离）和 p28（细胞间）由 1440nt 的亚基因组 RNA（sgRNA2）表达。在所有 Umbraviruses 中，sgRNA2 转录起始位点的上游都鉴定出一个高度保守的结构，其中包括一个 Carmovirus 保守序列，这表明它是通过 RdRp 介导的机制产生的。OPMV 还有第二个 1554nt 的 sgRNA1（sgRNA1），它刚好在一个典型的外切核酸酶抗性序列（xrRNA）的下游开始。sgRNA1 编码一个 30kDa 的蛋白质，与 p28 框内，不能在其他 Umbraviruses 中合成。消除 sgRNA1 或截断 p30 开放阅读框（ORF）而不显著减少 OPMV gRNA 的积累，表明该蛋白具有功能作用。OPMV 的 652nt 3'非翻译区包含两个 3'帽非依赖性翻译增强子（3' CITEs），在其 3'端附近有一个 T 形结构（TSS），在中央区域有一个类似于翻译元件（BTE）。只有 BTE 在包含 gRNA 或 sgRNA2 5'序列的荧光素酶报告基因构建体中具有功能，这与 Umbravirus 3' CITEs 在先前的研究中使用的方式不同。与大多数 3' CITEs 一样，OPMV 的 BTE 通过远距离 RNA-RNA 相互作用与 5'端连接。对 14 个 BTE 的分析揭示了除先前鉴定的 17 个核苷酸保守序列之外的其他保守序列和结构特征。（OPMV）是 Umbravirus 家族的一员。我们确定 OPMV 积累了两个大小相似的亚基因组 RNA（sgRNAs），较小的已知编码来自重叠开放阅读框表达的蛋白质。稍大的 sgRNA1 的 5'端刚好在上游的一个先前预测的 xrRNA 位点，将该 sgRNA 鉴定为一个异常长的产物，由外切核酸酶修剪产生。尽管有四个 Umbraviruses 具有类似的预测 xrRNA 位点，但只有 OPMV 的 sgRNA1 可以编码 Umbravirus ORF4 的延伸产物。无法生成 sgRNA 或翻译该蛋白与 gRNA 积累减少有关。我们还对 OPMV 的 BTE 结构，即 3'帽非依赖性翻译增强子（3' CITE）进行了表征。与 OPMV BTE 的 13 个 BTE 的比较揭示了除 17 个核苷酸特征序列之外的额外序列相似性，以及以前在这些 3' CITE 中未识别到的保守结构特征。
