Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

中文翻译：

---

冠状病毒温度敏感突变体的分析揭示了大域和木瓜蛋白酶类蛋白酶之间的相互作用影响复制和发病机理。

对温度敏感（ts）突变病毒的分析是一种经典方法，可让研究人员识别参与病毒复制和发病机理的遗传基因座。在这里，我们报告的小鼠肝炎病毒（MHV）tsNC11 ts菌株的遗传分析，重点关注突变在非结构蛋白3（nsp3）的大结构域（MAC）和木瓜蛋白酶2（PLP2）域中的作用，是病毒复制复合体的组成部分。使用MHV反向遗传学，我们生成了一系列突变病毒，以定义大结构域和PLP2特异性突变对ts表型的贡献。病毒复制动力学和在许可和非许可温度下进行的铺板效率分析表明，对于ts表型，仅大结构域的变化既必要也是充分的。有趣的是 PLP2结构域中的突变与温度敏感性无关，但确实降低了宏结构域突变体回复的频率。免疫共沉淀研究与大结构域和PLP2之间的相互作用是一致的。对nsp3的宏结构域-PLP2部分的表达研究表明，ts突变增强了蛋白酶体介导的蛋白质降解。此外，我们发现在病毒感染期间，包含MAC和PLP2突变的复制酶蛋白在非许可温度下比野生型蛋白降解更快。重要的是，我们显示了巨域和PLP2突变病毒在体外触发了I型干扰素的产生，并在小鼠中被减弱，进一步突出了巨域-PLP2相互作用在病毒发病机理中的重要性。重要信息冠状病毒（CoV）是新兴的具有大流行潜力的人类和兽医病原体。尽管这些病毒已经对人类健康造成了确定和预测的威胁，但目前尚无批准的对策来控制人类中这些病毒的感染。病毒大域，去除蛋白质翻译后ADP-核糖基化的酶以及病毒多功能木瓜蛋白酶样蛋白酶，裂解多蛋白并通过去泛素化活性去除多聚泛素链的酶是两个重要的毒力因子。在这里，我们揭示了宏域和PLP2域之间的意外相互作用，这对于复制和拮抗宿主先天免疫应答非常重要。靶向这些酶的相互作用可能为治疗CoV疾病提供新的治疗机会。