Chen Jianbo, Noueiry Amine, Ahlquist Paul
Institute for Molecular Virology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706-1596, USA.
J Virol. 2003 Feb;77(4):2568-77. doi: 10.1128/jvi.77.4.2568-2577.2003.
The multidomain RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, plays key roles in assembly and function of the viral RNA replication complex. 1a, which encodes RNA capping and helicase-like domains, localizes to endoplasmic reticulum membranes, recruits BMV 2a polymerase and viral RNA templates, and forms membrane-bound, capsid-like spherules in which RNA replication occurs. cis-acting signals necessary and sufficient for RNA recruitment by 1a have been mapped in BMV genomic RNA2 and RNA3. Both signals comprise an extended stem-loop whose apex matches the conserved sequence and structure of the TPsiC stem-loop in tRNAs (box B). Mutations show that this box B motif is crucial to 1a responsiveness of wild-type RNA2 and RNA3. We report here that, unexpectedly, some chimeric mRNAs expressing the 2a polymerase open reading frame from RNA2 were recruited by 1a to the replication complex and served as templates for negative-strand RNA synthesis, despite lacking the normally essential, box B-containing 5' signal. Further studies showed that this template recruitment required high-efficiency translation of the RNA templates. Moreover, multiple small frameshifting insertion or deletion mutations throughout the N-terminal region of the open reading frame inhibited this template recruitment, while an in-frame insertion did not. Providing 2a in trans did not restore template recruitment of RNAs with frameshift mutations. Only those deletions in the N-terminal region of 2a that abolished 2a interaction with 1a abolished template recruitment of the RNA. These and other results indicate that this alternate pathway for 1a-dependent RNA recruitment involves 1a interaction with the translating mRNA via the 1a-interactive N-terminal region of the nascent 2a polypeptide. Interaction with nascent 2a also may be involved in 1a recruitment of 2a polymerase to membranes.
雀麦花叶病毒（BMV）是α病毒样超家族中的一种正链RNA病毒，其多结构域RNA复制蛋白1a在病毒RNA复制复合体的组装和功能中起关键作用。1a编码RNA封端和螺旋酶样结构域，定位于内质网，招募BMV 2a聚合酶和病毒RNA模板，并形成膜结合的、衣壳样小球，RNA复制在其中发生。在BMV基因组RNA2和RNA3中已定位了1a招募RNA所必需且充分的顺式作用信号。这两个信号都包含一个延伸的茎环，其顶端与tRNA中TPsiC茎环的保守序列和结构相匹配（B框）。突变表明，这个B框基序对于野生型RNA2和RNA3的1a反应性至关重要。我们在此报告，出乎意料的是，一些从RNA2表达2a聚合酶开放阅读框的嵌合mRNA被1a招募到复制复合体，并作为负链RNA合成的模板，尽管它们缺乏通常必不可少的、含B框的5'信号。进一步研究表明，这种模板招募需要RNA模板的高效翻译。此外，开放阅读框N端区域的多个小移码插入或缺失突变抑制了这种模板招募，而框内插入则没有。反式提供2a并不能恢复具有移码突变的RNA的模板招募。只有2a N端区域那些消除了2a与1a相互作用的缺失才消除了RNA的模板招募。这些以及其他结果表明，这种依赖1a的RNA招募的替代途径涉及1a通过新生2a多肽的1a相互作用N端区域与正在翻译的mRNA相互作用。与新生2a的相互作用也可能参与1a将2a聚合酶招募到膜上。
