Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication.
Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication.
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DOI：
10.1128/jvi.00469-16
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发表时间：
2016-08-01
影响因子：
5.4
通讯作者：
Stonehouse NJ
中科院分区：
文献类型：
--
作者：
Herod MR;Ferrer-Orta C;Loundras EA;Ward JC;Verdaguer N;Rowlands DJ;Stonehouse NJ
PicorNaviridae是一个正义RNA病毒大家族，包含许多人类和动物病原体，包括口蹄疫病毒(FMDV)。小核糖核酸病毒复制复合体由涉及多种病毒和宿主细胞因子的蛋白质-蛋白质和蛋白质-RNA相互作用的协调网络组成。该复合体中的许多蛋白质在病毒RNA复制中具有多种作用，其中一些蛋白质可以反式提供(即通过从单独的RNA分子表达)，而另一些蛋白质是顺式需要的(即通过模板RNA分子表达)。体外研究表明，RNA依赖的RNA聚合酶(RdRp)3D的多个副本参与了病毒复制复合体。然而，目前还不清楚是否所有这些分子都具有催化活性，或者它们还提供什么其他功能(S)。在这项研究中，我们的目标是区分催化活性的3D分子和那些建立复制复合体的分子。我们报道了3D的一种新的非酶顺式作用功能，它是病毒基因组复制所必需的。使用FMDV复制子进行互补实验，我们的数据表明3D的这种顺式作用不同于以反式作用为主的催化活性。免疫荧光研究表明，顺式和反式作用的3D分子都定位在同一细胞隔间。然而，我们的遗传和结构数据表明，3D在顺式结构中与RNA茎环相互作用，这是病毒RNA复制所必需的。这项研究确定了微小核糖核酸病毒复制复合体结构-功能的一个以前未描述的方面，以及进一步探索这种相互作用的重要方法。口蹄疫病毒(FMDV)是引起口蹄疫的重要动物病原体。该疾病在世界许多地区流行，在牲畜中暴发，造成重大经济损失。病毒基因组的传播发生在复制复合体中，了解这一过程有助于开发新的治疗策略。许多参与复制的非结构蛋白在病毒生命周期中具有多种功能，其中一些可以从单独的基因组(即反式)提供给复制复合体，而另一些必须来自模板(即顺式)。在这里，我们提出了对RNA依赖的RNA聚合酶3D的顺式和反式活性的分析。我们展示了3D在复制中的一个新的顺式作用。我们的数据表明，这种作用不同于它的酶功能，需要与病毒基因组相互作用。我们的数据进一步加深了对这种重要病原体基因组复制的理解。
The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in trans) while others must originate from the template (i.e., in cis). Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.