Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors.

The Journal of Biological Chemistry Pub Date : 2023-11-01 Epub Date: 2023-09-15 DOI:10.1016/j.jbc.2023.105258
Grace Campagnola, Olve Peersen
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Abstract

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

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脊髓灰质炎病毒3Cpro多蛋白前体的共折叠和RNA激活。
正链RNA病毒使用长的开放阅读框来表达大的多蛋白,这些多蛋白被病毒蛋白酶加工成单个蛋白质。多蛋白加工受到高度调节,产生与完全加工的蛋白质具有不同功能的中间物种,增加了紧凑病毒基因组的生物化学多样性,同时也带来了蛋白质必须在多种情况下保持稳定折叠的挑战。我们使用圆二色光谱和单分子显微镜检查了脊髓灰质炎病毒P3区蛋白的溶液结构和自缔合,该蛋白由膜结合3A、RNA引发3B(VPg)、3Cpro蛋白酶和3Dpol RNA依赖性RNA聚合酶蛋白组成。我们的数据表明,3ABC片段内的共折叠相互作用稳定了3C蛋白酶区域的构象状态,这种稳定需要全长3A和3B蛋白。酶活性测定表明,3ABC也是一种活性蛋白酶,其切割肽底物的速率与3Cpro相当。RNA的加入刺激了较大多蛋白底物的切割,在化学计量量的病毒cre RNA存在下,3ABCpro的活性比3Cpro高20倍。数据表明,3ABC区域内的共折叠导致蛋白酶可以通过定位到病毒复制中心内的特定RNA元件而被高度活化到某些切割位点,从而提供了调节病毒多蛋白加工的机制。
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