In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.
In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.
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病毒相关 RNA I (VAI RNA) 的体外分析：VAI RNA 突变体对双链 RNA 激活蛋白激酶 PKR 的抑制与体内表型和中心结构域的结构完整性相关。
DOI：
10.1128/jvi.68.7.4137-4151.1994
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发表时间：
1994
影响因子：
5.4
通讯作者：
Thimmapaya,B
中科院分区：
文献类型：
--
作者：
Ghadge,GD;Malhotra,P;Furtado,MR;Dhar,R;Thimmapaya,B
腺病毒使用病毒编码的病毒相关 RNA (VAI RNA)，通过阻断干扰素诱导的双链 RNA 激活蛋白激酶 PKR 的激活来防御细胞抗病毒反应。 VAI RNA 的结构由两个长的、不完全碱基配对的双链体区域组成，由中心的复杂短茎环（称为中心结构域）连接。通过使用一系列 VAI RNA 基因中具有接头扫描突变的腺病毒突变体，我们最近表明 VAI RNA 分子功能所需的关键元件位于中心结构域，并且中心结构域的这些相同元件也参与与 PKR 的结合。在病毒感染的细胞中，VAI RNA 与潜在激酶相互作用，后者与核糖体结合；这种相互作用发生在复杂的环境中。为了更全面地了解结构和功能之间的关系，并确定这些突变体的体内表型是否可以在体外复制，我们现在分析了这些突变体 VAI 等位基因，以确定它们阻止 HeLa 细胞部分纯化的 PKR 激活的能力。我们还通过实验推导了这些突变体的结构，并将结构与功能相关联。无一例外的是，当中央结构域的短茎环结构受到干扰时，突变体无法抑制 PKR。分子中心结构域或长双链体区域其他位置的结构破坏对于体外功能并不有害。因此，这些结果支持了我们之前的发现，并强调了 VAI RNA 中心域中存在的元件对其功能的重要性。我们的结果还表明 PKR 和 VAI RNA 之间的相互作用涉及中心结构域中精确的二级（和三级）结构。有人认为，VAI RNA 不会激活病毒感染细胞中的 PKR，因为不完美碱基配对的长双链体区域存在错配。我们构建了突变的 VAI 基因，其中不完全碱基配对的双链体区域被转化为完美碱基配对的区域，并在体外测定了 PKR 的激活。与野生型VAI RNA一样，这些突变体未能在体外激活PKR，但它们能够比野生型更好地阻断PKR的激活。这些结果表明，VAI RNA 激活 PKR 的失败并不是长双链体区域错配的结果。（摘要截断为 400 字）
Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experimentally and correlated the structure with function. Without exception, when the structure of the short stem-loop of the central domain was perturbed, the mutants failed to inhibit PKR. Structural disruptions elsewhere in the central domain or in the long duplex regions of the molecule were not deleterious for in vitro function. Thus, these results support our previous findings and underscore the importance of the elements present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA involves a precise secondary (and tertiary) structure in the central domain. It has been suggested that VAI RNA does not activate PKR in virus-infected cells because of mismatches in the imperfectly base-paired long duplex regions. We constructed mutant VAI genes in which the imperfectly base-paired duplex regions were converted to perfectly base-paired regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, while they were able to block the activation of PKR better than did the wild type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions.(ABSTRACT TRUNCATED AT 400 WORDS)
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