Wahid Ahmed M, Coventry Veronica K, Conn Graeme L
Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester, M1 7DN.
Nucleic Acids Res. 2009 Sep;37(17):5830-7. doi: 10.1093/nar/gkp595. Epub 2009 Jul 27.
VA RNA(I) is a non-coding adenoviral transcript that counteracts the host cell anti-viral defenses such as immune responses mediated via PKR. We investigated potential alternate secondary structure conformations within the PKR-binding domain of VA RNA(I) using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNA(I) apical stem can adopt two different conformations and that it exists as a mixed population of these two structures. In contrast, in two sequence variants we designed to eliminate one of the possible structures, while leaving the other intact, each formed a unique secondary structure. This clarification of the apical stem pairing also suggests a small alteration to the apical stem-loop secondary structure. The relative ability of the two apical stem conformations to bind PKR and inhibit kinase activity was measured by isothermal titration calorimetry and PKR autophosphorylation inhibition assay. We found that the two sequence variants displayed markedly different activities, with one being a significantly poorer binder and inhibitor of PKR. Whether the presence of the VA RNA(I) conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation.
VA RNA（I）是一种非编码腺病毒转录本，可对抗宿主细胞的抗病毒防御机制，如通过PKR介导的免疫反应。我们使用定点诱变、RNA紫外熔解分析和酶促RNA二级结构探测，研究了VA RNA（I）的PKR结合域内潜在的替代二级结构构象。后者的数据清楚地表明，野生型VA RNA（I）顶端茎可以采用两种不同的构象，并且它以这两种结构的混合群体形式存在。相比之下，在我们设计的两个序列变体中，消除了其中一种可能的结构，而另一种保持完整，每个都形成了独特的二级结构。顶端茎配对的这种阐明也表明顶端茎环二级结构有微小变化。通过等温滴定量热法和PKR自磷酸化抑制试验，测定了两种顶端茎构象结合PKR和抑制激酶活性的相对能力。我们发现这两个序列变体表现出明显不同的活性，其中一个作为PKR的结合剂和抑制剂明显较差。具有降低的PKR抑制活性的VA RNA（I）构象的存在是否对细胞中的病毒在某些其他功能上直接有益，需要进一步研究。
