Department of Microbiology and Immunology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, USA.
Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA.
J Virol. 2020 May 18;94(11). doi: 10.1128/JVI.01734-19.
Coronaviruses express a multifunctional papain-like protease, termed papain-like protease 2 (PLP2). PLP2 acts as a protease that cleaves the viral replicase polyprotein and as a deubiquitinating (DUB) enzyme which removes ubiquitin (Ub) moieties from ubiquitin-conjugated proteins. Previous studies implicated PLP2/DUB activity as a negative regulator of the host interferon (IFN) response, but the role of DUB activity during virus infection was unknown. Here, we used X-ray structure-guided mutagenesis and functional studies to identify amino acid substitutions within the ubiquitin-binding surface of PLP2 that reduced DUB activity without affecting polyprotein processing activity. We engineered a DUB mutation (Asp1772 to Ala) into a murine coronavirus and evaluated the replication and pathogenesis of the DUB mutant virus (DUBmut) in cultured macrophages and in mice. We found that the DUBmut virus replicates similarly to the wild-type (WT) virus in cultured cells, but the DUBmut virus activates an IFN response at earlier times compared to the wild-type virus infection in macrophages, consistent with DUB activity negatively regulating the IFN response. We compared the pathogenesis of the DUBmut virus to that of the wild-type virus and found that the DUBmut-infected mice had a statistically significant reduction ( < 0.05) in viral titer in liver and spleen at day 5 postinfection (d p.i.), although both wild-type and DUBmut virus infections resulted in similar liver pathology. Overall, this study demonstrates that structure-guided mutagenesis aids the identification of critical determinants of the PLP2-ubiquitin complex and that PLP2/DUB activity plays a role as an interferon antagonist in coronavirus pathogenesis. Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.
冠状病毒表达一种多功能的木瓜蛋白酶样蛋白酶，称为木瓜蛋白酶样蛋白酶 2（PLP2）。PLP2 作为一种蛋白酶，可切割病毒复制酶多蛋白，作为去泛素化（DUB）酶，可从泛素化缀合蛋白上去除泛素（Ub）部分。先前的研究表明，PLP2/DUB 活性是宿主干扰素（IFN）反应的负调节剂，但病毒感染期间 DUB 活性的作用尚不清楚。在这里，我们使用 X 射线结构引导的诱变和功能研究来鉴定 PLP2 中泛素结合表面内的氨基酸取代，这些取代降低了 DUB 活性而不影响多蛋白加工活性。我们将 DUB 突变（天冬氨酸 1772 突变为丙氨酸）引入鼠冠状病毒中，并在培养的巨噬细胞中和小鼠中评估 DUB 突变病毒（DUBmut）的复制和发病机制。我们发现，DUBmut 病毒在培养细胞中的复制与野生型（WT）病毒相似，但与 WT 病毒感染巨噬细胞相比，DUBmut 病毒更早地激活 IFN 反应，这与 DUB 活性负调控 IFN 反应一致。我们比较了 DUBmut 病毒和 WT 病毒的发病机制，发现 DUBmut 感染的小鼠在感染后 5 天（d p.i.）肝和脾中的病毒滴度有统计学意义的降低（<0.05），尽管 WT 和 DUBmut 病毒感染均导致相似的肝病理。总体而言，这项研究表明，结构引导的诱变有助于确定 PLP2-泛素复合物的关键决定因素，并且 PLP2/DUB 活性在冠状病毒发病机制中作为干扰素拮抗剂发挥作用。冠状病毒通过编码在病毒复制中起作用的多功能蛋白并修饰宿主环境来解除先天免疫反应，从而采用遗传经济性。冠状病毒的木瓜蛋白酶样蛋白酶 2（PLP2）结构域具有蛋白酶活性，可切割病毒复制酶多蛋白，还具有 DUB 活性（使用相同的催化残基从修饰的底物上去除泛素/泛素样分子）。为了将 DUB 活性与蛋白酶活性分开，我们采用了一种结构引导的诱变方法，并鉴定了对泛素结合很重要的残基。我们发现，突变泛素结合残基会导致具有降低的 DUB 活性但保留蛋白酶活性的 PLP2。我们设计了一种表达 DUB 突变的重组鼠冠状病毒，并表明 DUB 突变病毒在巨噬细胞中更早地激活 I 型干扰素反应，并在小鼠中表现出复制减少。这项研究的结果表明，PLP2/DUB 是一种干扰素拮抗剂，也是冠状病毒的毒力特征。
