A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPase Causes Conditional Defects in Encapsidation and Uncoating.
A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPase Causes Conditional Defects in Encapsidation and Uncoating.
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DOI：
10.1128/jvi.02877-15
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发表时间：
2016-07-15
影响因子：
5.4
通讯作者：
Paul AV
中科院分区：
文献类型：
--
作者：
Asare E;Mugavero J;Jiang P;Wimmer E;Paul AV
C簇肠道病毒衣壳化的特异性依赖于衣壳蛋白和非结构蛋白2CATALYST之间的相互作用。具体地，在嵌合病毒的情况下，脊髓灰质炎病毒2CATALYST的残基N252与柯萨奇病毒A20的VP 3相互作用。脊髓灰质炎病毒2CATALYST在RNA复制和降解中起重要作用。在这项研究中，我们在N252附近的2CATALYST中搜索了额外的位点，这些位点是进行腺苷酸化所必需的。因此，通过组合三重和单丙氨酸突变来分析与N252相邻的区段以鉴定功能所需的残基。两个三重丙氨酸突变体表现出RNA复制缺陷。剩余的两个突变，位于二级结构中的预测三维模型的2CATALYST，造成致命的生长表型。大多数单丙氨酸突变体，衍生自致死性变体，是准感染性的，并产生具有野生型（wt）或温度敏感型（ts）生长表型的变体，或由于RNA复制缺陷而具有致死性生长表型。K259 A突变，映射到2CATALYST预测结构中的α螺旋，导致了冷敏感病毒。体内蛋白质合成和病毒产生在33°C下相对于wt显著延迟，表明未包被的缺陷。用报告病毒进行的研究表明，该突变体在33°C下也存在糖苷化缺陷。细胞成像证实，相对于wt.综上所述，我们首次将2CATALYST（K259 A）中的冷敏感性包被缺陷与病毒颗粒在下一个感染周期中在33°C下的脱包被延迟联系起来。重要性肠道病毒的形态发生，包括新产生的病毒体RNA的降解，是一个仍然知之甚少的过程。阐明这一过程是重要的未来药物开发的各种疾病所造成的这些代理。我们以前已经表明，脊髓灰质炎病毒和C-簇柯萨奇病毒，这是肠道病毒的原型，是依赖于衣壳蛋白与多功能非结构蛋白2CATALYST的相互作用的特异性。在这项研究中，我们已经搜索了脊髓灰质炎病毒2CATALYST的残留物，附近的一个假定的capaly的相互作用的网站，重要的是，pH2.5。一种不寻常的2CATALYST冷敏感突变体在37°C下具有包被缺陷，随后在33°C下的下一个感染周期中脱膜。这些研究不仅揭示了一个新的网站，在2CATALYST，参与了磷脂化，但也确定了磷脂化和脱壳之间的联系。
The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2CATPase. In particular, residue N252 of poliovirus 2CATPase interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2CATPase has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2CATPase, near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2CATPase, caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2CATPase, resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2CATPase (K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection. IMPORTANCE Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2CATPase. In this study, we have searched for residues in poliovirus 2CATPase, near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2CATPase possessed a defect in encapsidation at 37°C and subsequently in uncoating during the next cycle of infection at 33°C. These studies not only reveal a new site in 2CATPase that is involved in encapsidation but also identify a link between encapsidation and uncoating.