THE ANTIVIRAL COMPOUND ENVIROXIME TARGETS THE 3A CODING REGION OF RHINOVIRUS AND POLIOVIRUS
THE ANTIVIRAL COMPOUND ENVIROXIME TARGETS THE 3A CODING REGION OF RHINOVIRUS AND POLIOVIRUS
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DOI：
10.1128/jvi.69.7.4189-4197.1995
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发表时间：
1995-07-01
影响因子：
5.4
通讯作者：
VANCE, LM
中科院分区：
文献类型：
--
作者：
HEINZ, BA;VANCE, LM
Enviroxime是一种抗病毒化合物，可抑制鼻病毒和肠道病毒的复制。我们以脊髓灰质炎病毒1型和人鼻病毒14型为模型系统，探讨了环磷肟的作用机制。通过改变药物添加到病毒感染细胞的时间，我们确定了enviroxime可以在感染后几小时添加，而没有明显的抑制损失。这表明该药物靶向参与RNA复制或蛋白质加工的步骤。为了确定这一目标，我们绘制了23个独立的突变突变体，可以在每毫升1微克的enviroxime的存在下繁殖。这些突变体中的每一个都含有改变3A编码区中的一个氨基酸的单核苷酸取代。使用cDNA克隆的寡核苷酸定向诱变，我们已经证实这些单氨基酸取代足以赋予抗性表型。此外，我们进行了两个实验，以支持假设，enviroxime抑制3A功能。首先，我们通过对脊髓灰质炎病毒感染细胞的RNA进行斑点印迹分析，确定恩伏肟优先抑制病毒正链的合成。第二，我们证明了enviroxime抑制正链RNA合成的起始，通过在脊髓灰质炎病毒粗复制复合物中向3AB添加[P-32]尿苷来测量。据我们所知，这是3A可以被抗病毒药物靶向的第一个证据。我们预计，enviroxime将是一个有用的工具，调查的自然功能的3A蛋白。
Enviroxime is an antiviral compound that inhibits the replication of rhinoviruses and enteroviruses. We have explored the mechanism of action of enviroxime by using poliovirus type 1 and human rhinovirus type 14 as model systems. By varying the time of drug addition to virus-infected cells, we determined that enviroxime could be added several hours postinfection without significant loss of inhibition. This suggested that the drug targeted a step involved in RNA replication or protein processing. To identify this target, we mapped 23 independent mutations in mutants that could multiply in the presence of 1 mu g of enviroxime per mi. Each of these mutants contained a single nucleotide substitution that altered one amino acid in the 3A coding region. Using oligonucleotide-directed mutagenesis of cDNA clones, we have confirmed that these single-amino-acid substitutions are sufficient to confer the resistance phenotype. In addition, we conducted two experiments to support the hypothesis that enviroxime inhibits a 3A function. First, we determined by dot blot analysis of RNA from poliovirus-infected cells that enviroxime preferentially inhibits synthesis of the viral plus strand. Second, we demonstrated that enviroxime inhibits the initiation of plus-strand RNA synthesis as measured by the addition of [P-32]uridine to 3AB in poliovirus crude replication complexes. To our knowledge, this is the first evidence that 3A can be targeted by antiviral drugs. We anticipate that enviroxime will be a useful tool for investigating the natural function of the 3A protein.