Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3′ CITE
Opium Poppy Mosaic Virus Has an Xrn-Resistant, Translated Subgenomic RNA and a BTE 3′ CITE
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DOI：
10.1128/jvi.02109-20
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发表时间：
2021-05-01
影响因子：
5.4
通讯作者：
Simon, Anne E.
中科院分区：
文献类型：
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作者：
Ilyas, Muhammad;Du, Zhiyou;Simon, Anne E.
罂粟花叶病毒(OPMV)是近年来在番茄病毒科新发现的一种枝状病毒。OPMV具有4,241个核苷酸(NT)的正义基因组RNA(GRNA)，表达复制蛋白p35和p35延伸产物p98，即RNA依赖的RNA聚合酶(RdRp)。运动蛋白p27(长距离)和p28(细胞间)从1,440个核苷酸的亚基因组RNA(SgRNA2)表达。在所有真菌病毒中，在sgRNA2转录起始点的上游都发现了一个高度保守的结构，其中包括一个卡莫病毒共同序列，表明是由RdRp介导的机制产生的。OPMV还有第二个sgRNA，长1,554个核苷酸(SgRNA1)，恰好开始于一个典型的外切核酸酶抗性序列(XrRNAD)的下游。SgRNA1在体外编码一个30 kDa的蛋白，与p28相框，不能在其他枝状病毒中合成。在不影响p28的情况下，消除sgRNA1或截短P30开放阅读框架(ORF)可显著减少OPMV gRNA的积累，这表明该蛋白具有功能作用。OPMV的652-nt3‘非翻译区含有两个3’端非依赖帽的翻译增强子(3‘CITES)，在其3’端附近有一个T型结构(TSS)，在中心区有一个大麦黄矮病毒样翻译元件(BTE)。在体内，只有BTE在含有gRNA或sgRNA2 5‘序列的荧光素酶报告结构中具有功能，这与先前研究中使用的umbraVirus 3’Cite不同。与大多数3‘端CITE类似，OPMV BTE通过长距离RNA-RNA相互作用连接到5’端。对14个BTE序列的分析揭示了比已知的17个核苷酸保守序列更多的保守序列和结构特征。重要的是鸦片罂粟花叶病毒(OPMV)是番茄病毒科的一种枝状病毒。我们确定OPMV积累了两个大小相似的亚基因组RNA(SgRNAs)，其中较小的已知编码来自重叠开放阅读框架的蛋白质。稍大的sgRNA1在先前预测的xrRNAD位点上游有一个5‘端，证明该sgRNA是由外切核酸酶修剪产生的异常长的产物。虽然四种umbra病毒有相似的xrRNAD预测位点，但只有OPMV的sgRNA1可以编码一种作为umbra病毒ORF4延伸产物的蛋白质。不能产生sgRNA或翻译这种蛋白质与体内gRNA积累减少有关。我们还对OPMV BTE结构进行了表征，它是一种3‘帽非依赖性翻译增强子(3’Cite)。将13个BTE与OPMV BTE进行比较，发现了17-nT特征序列之外的额外序列相似性，以及以前在这些3‘CITE中未发现的保守结构特征。
Opium poppy mosaic virus (OPMV) is a recently discovered umbravirus in the family Tombusviridae. 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 (xrRNAD). sgRNA1 codes for a 30-kDa protein in vitro 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 Barley yellow dwarf virus-like translation element (BTE) in the central region. Only the BTE is functional in luciferase reporter constructs containing gRNA or sgRNA2 5' sequences in vivo, 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.IMPORTANCE Opium poppy mosaic virus (OPMV) is an umbravirus in the family Tombusviridae. 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 xrRNAD site, identifying this sgRNA as an unusually long product produced by exoribonuclease trimming. Although four umbraviruses have similar predicted xrRNAD 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 in vivo. 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.