Brome mosaic virus protein 1a recruits viral RNA2 to RNA replication through a 5′ proximal RNA2 signal
Brome mosaic virus protein 1a recruits viral RNA2 to RNA replication through a 5′ proximal RNA2 signal
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DOI：
10.1128/jvi.75.7.3207-3219.2001
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发表时间：
2001-04-01
影响因子：
5.4
通讯作者：
Ahlquist, P
中科院分区：
文献类型：
--
作者：
Chen, JB;Noueiry, A;Ahlquist, P
溴花叶病毒（BMV）是甲病毒超家族中的一种正链RNA病毒，编码两种RNA复制因子。膜相关la蛋白含有解旋酶样结构域和RNA旋盖功能，la靶向膜的2a含有中心聚合酶样结构域，在缺乏Za和RNA复制的情况下，la通过BMV基因组RNA3的基因间复制信号来稳定RNA3并诱导RNA3与细胞膜结合，多重结果表明，1a诱导的RNA3稳定反映了招募RNA3模板参与复制的相互作用。为了确定la是否对另一个Bhn RNA复制模板有类似的作用，我们构建了一个表达briv基因组RNA2的质粒，在体内表达la和2a时复制体内表达的RNA2模板，在2a缺失的情况下，la稳定RNA2并诱导RNA2与膜结合。缺失分析表明，la诱导的RNA2的膜结合是由RNA2的5‘-近三分之一的序列介导的，RNA2的5’未翻译区足以将la诱导的膜结合赋予非病毒RNA。然而，Za开放阅读框n端区域的序列增强了RNA2和嵌合RNA的la响应性，一个对RNA2复制重要的5'端RNA2茎环对于la诱导的RNA2膜结合至关重要，并且与la响应的RNA3基因间区域一样，包含与宿主trna的T PsiC茎环对应的必需box B基序。各种RNA2突变体的la诱导的膜关联水平与其作为复制模板的能力密切相关，这些结果支持并扩展了a诱导的BMV RNA稳定和膜关联反映了病毒RNA复制的早期，1a介导的步骤。
Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated la protein contains a helicase-like domain and RNA capping functions, 2a, which is targeted to membranes by la, contains a central polymerase-like domain, In the absence of Za and RNA replication, la acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane, Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication, To determine if la had similar effects on another Bhn RNA replication template, we constructed a plasmid expressing BR IV genomic RNA2 in vivo, In vivo-expressed RNA2 templates were replicated upon expression of la and 2a, In the absence of 2a, la stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that la-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2, The RNA2 5' untranslated region was sufficient to confer la-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the Za open reading frame enhanced la responsiveness of RNA2 and a chimeric RNA, A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for la-induced membrane association of RNA2 and, like the la-responsive RNA3 intergenic region, contained a required box B motif corresponding to the T PsiC stem-loop of host tRNAs, The Level of la-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates, These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.