Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways.
Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways.
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DOI：
10.1371/journal.ppat.1006666
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发表时间：
2017-10
期刊：
影响因子：
6.7
通讯作者：
Stonehouse NJ
中科院分区：
文献类型：
--
作者：
Herod MR;Gold S;Lasecka-Dykes L;Wright C;Ward JC;McLean TC;Forrest S;Jackson T;Tuthill TJ;Rowlands DJ;Stonehouse NJ
小核糖核酸病毒的RNA基因组被翻译成单个多蛋白，其随后被切割成结构和非结构蛋白产物。对于遗传经济，蛋白质和加工中间体已经进化为执行不同的功能。小核糖核酸病毒前体蛋白P3被切割以产生膜结合3A、引物肽3B、蛋白酶3Cpro和聚合酶3Dpol。口蹄疫病毒（FMDV）编码3个相似的3B拷贝（3B1 - 3），这为研究3B蛋白在生物体内的作用提供了一个方便的天然系统。使用复制子系统，我们通过基因缺失或功能失活证实了3B的每个拷贝似乎独立地起作用以引发FMDV RNA复制。然而，我们还表明，3B3的缺失会阻止复制，这可以通过在3B2的C-末端引入突变来逆转，这些突变恢复了3B3 - 3C切割位点的天然序列。体外翻译研究表明，与3B3删除的前体被迅速切割，产生3CD，但没有聚合酶，3Dpol，被检测到。使用携带不同失活突变的可区分复制子的互补测定显示，具有3Dpol内突变的复制子可以通过衍生自"辅助"复制子（在3B的所有三个拷贝中掺入失活突变）的3Dpol回收。然而，当天然3B-3C切割位点在"辅助"复制子中改变时，没有观察到互补，再次表明在该位置的加工异常阻止了3Dpol的产生。当将影响多蛋白加工的突变引入感染性克隆时，回收了活病毒，但这些病毒在3B-3C切割位点获得了补偿突变。当在体外加工测定中分析时，这些突变显示出恢复野生型加工特征。总的来说，这项研究证明了小引物肽3B3的双重功能作用，进一步强调了小核糖核酸病毒如何增加遗传经济。正链RNA病毒通常以小的基因组大小为代表。为了弥补这一局限性，他们已经发展出了从一个小基因组中获得功能的方法，从而增加了“遗传经济”。在RNA病毒的小核糖核酸病毒家族中，病毒蛋白通过一系列瞬时前体产生，据信这些前体具有病毒复制所需的功能，这些功能是完全加工的蛋白质的功能之外的。在这份报告中，我们表明，从口蹄疫病毒，一种经济上重要的动物病原体的小3B3蛋白，是在指导P3多聚蛋白的加工途径，以促进释放的病毒聚合酶的关键。这是除了3B3作为复制引物的作用之外的作用，并且独立于3B3作为复制引物的作用。这项研究阐明了一个小病毒蛋白中的多种功能如何控制病毒复制，并展示了小核糖核酸病毒遗传经济的新水平。此外，提供反式"帮助"以辅助复制的系统具有被开发用于生产减毒疫苗的潜力（例如使用"辅助"细胞系）。我们现在正在研究多蛋白加工边界的突变如何用于生产减毒疫苗。
The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from “helper” replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the “helper” replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy. Positive-strand RNA viruses are generally typified by a small genome size. To compensate for this limitation they have evolved ways of multiplying the functions achievable from a small genome, thus increasing ‘genetic economy’. In the picornavirus family of RNA viruses the viral proteins are produced via a series of transient precursors, which are believed to possess functions required for virus replication which are additional to those of the fully processed proteins. In this report, we show that the small 3B3 protein from foot-and-mouth disease virus, an economically important animal pathogen, is key in directing the processing pathways of the P3 polyprotein to facilitate release of the viral polymerase. This is in addition to, and independent from, the role of 3B3 as a primer for replication. This study exemplifies how multiple functions within one small viral protein can control viral replication and demonstrates a new level of genetic economy in the picornaviruses. Furthermore, systems that provide ‘help’ in trans to assist replication have the potential to be exploited for the production of attenuated vaccines (e.g. using “helper’ cell lines). We are now in the process of investigating how mutations at polyprotein processing boundaries may be used for producing attenuated vaccines.
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