May Jared, Johnson Philip, Saleem Huma, Simon Anne E
Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, College Park, Maryland, USA.
Department of Cell Biology and Molecular Genetics, University of Maryland-College Park, College Park, Maryland, USA
J Virol. 2017 Mar 29;91(8). doi: 10.1128/JVI.02421-16. Print 2017 Apr 15.
To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5' cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both and An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation. Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein and Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common.
为了最大限度地发挥病毒基因组的编码潜力，内部核糖体进入位点（IRES）可用于绕过对5′帽和一些/所有相关翻译起始因子的传统需求。尽管病毒IRES通常包含高阶RNA结构，但在芜菁皱缩病毒（TCV）外壳蛋白（CP）开放阅读框（ORF）上游紧邻的约84个核苷酸（nt）的非结构化序列已被发现可促进CP从基因组RNA（gRNA）进行内部表达，无论是在体内还是体外。使用RNA折叠算法预测不存在广泛的RNA结构，并通过引物延伸分析的选择性2′-羟基酰化（SHAPE）RNA结构探测进行了确认。使用TCV gRNA和报告基因构建体对IRES区域进行分析，未发现任何序列特异性元件，反而表明整体缺乏结构是IRES活性的一个重要特征。CP IRES富含A，与方向无关，并且在同一属的病毒中高度保守。该IRES的活性依赖于eIF4G，但不依赖于eIF4E。在没有可检测到的TCV亚基因组RNA的情况下，CP积累水平较低，这强烈表明IRES在gRNA中具有活性。由于TCV CP还充当病毒沉默抑制因子，因此CP从病毒gRNA的早期翻译可能对于对抗宿主防御很重要。细胞mRNA IRES也缺乏广泛的RNA结构或序列保守性，这表明这种病毒IRES和细胞IRES可能具有相似的内部翻译起始策略。不依赖帽的翻译是正链单链RNA病毒绕过宿主细胞对5′帽结构需求的常见策略。一般而言，病毒IRES包含对活性至关重要的广泛二级结构。相比之下，我们证明病毒RNA中一个缺乏广泛二级结构的区域具有IRES活性，并产生低水平的病毒外壳蛋白，无论是在体内还是体外。我们的发现可能适用于同样很少或没有共同序列/结构的细胞mRNA IRES。
