Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Virol. 2024 Nov 19;98(11):e0059024. doi: 10.1128/jvi.00590-24. Epub 2024 Oct 29.
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world's population. Lytic HCMV replication in immunocompromised individuals or neonates can lead to severe disease in multiple organ systems and even death. The establishment of lytic replication is driven by the first viral proteins expressed upon infection, the immediate early proteins, which play a key role in creating an intracellular environment conducive to virus replication. Two immediate early proteins, the functional orthologs pTRS1 and pIRS1, stimulate immediate early gene expression by suppressing antiviral PKR/eIF2α signaling and enhance the translation of viral mRNAs independent of PKR antagonism. To better understand the molecular functions of pTRS1, we used proximity labeling proteomics to identify proteins that interact with pTRS1 in infected cells. Multiple novel host and viral interactors were identified, including the catalytic subunits of the protein phosphatase 1 (PP1) holoenzyme. Mutations to a PP1 catalytic subunit known to disrupt binding to PP1 regulatory subunits decreased binding to pTRS1. pTRS1 immune complexes contained phosphatase activity, and inhibition of phosphatase activity in transfected or infected cells reversed the ability of pTRS1 to inhibit the antiviral kinase PKR. Depletion of individual PP1 catalytic subunits decreased virus replication and increased the phosphorylation of the PKR substrate eIF2α. Taken together, our data suggest potential novel functions for pTRS1 and define a novel role for PP1 as an antagonist of the antiviral PKR/eIF2α signaling axis during HCMV infection.IMPORTANCEThe human cytomegalovirus (HCMV) pTRS1 and pIRS1 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining the molecular functions of pTRS1/pIRS1 is important for understanding the molecular events controlling HCMV replication and viral disease. These data provide new insights into potential pTRS1 functional roles, providing a starting point for others to understand new features of infected cell biology. Another important result of this study is the finding that specific protein phosphatase 1 (PP1) regulatory subunits are required to suppress PKR/eIF2α signaling, a critical cellular innate immune defense to viral infection. These data lay the groundwork for future efforts to discover therapeutics that disrupt pTRS1 interaction with PP1 allowing cellular defenses to limit HCMV replication and disease.
人巨细胞病毒（HCMV）是一种普遍存在的病原体，感染了世界上大多数人口。在免疫功能低下的个体或新生儿中，溶细胞 HCMV 复制可导致多器官系统严重疾病，甚至死亡。溶细胞复制的建立是由感染后第一个表达的病毒蛋白，即早期即刻蛋白驱动的，这些蛋白在创造有利于病毒复制的细胞内环境中起着关键作用。两种早期即刻蛋白，功能同源物 pTRS1 和 pIRS1，通过抑制抗病毒 PKR/eIF2α 信号来刺激早期即刻基因表达，并增强病毒 mRNA 的翻译，而不依赖于 PKR 拮抗作用。为了更好地理解 pTRS1 的分子功能，我们使用邻近标记蛋白质组学来鉴定感染细胞中与 pTRS1 相互作用的蛋白质。鉴定出了多个新的宿主和病毒相互作用物，包括蛋白磷酸酶 1（PP1）全酶的催化亚基。已知破坏与 PP1 调节亚基结合的 PP1 催化亚基突变降低了与 pTRS1 的结合。pTRS1 免疫复合物含有磷酸酶活性，并且在转染或感染的细胞中抑制磷酸酶活性可逆转 pTRS1 抑制抗病毒激酶 PKR 的能力。单个 PP1 催化亚基的耗竭会降低病毒复制并增加 PKR 底物 eIF2α 的磷酸化。总之，我们的数据表明 pTRS1 可能具有新的功能，并定义了 PP1 作为 HCMV 感染期间抗病毒 PKR/eIF2α 信号轴的拮抗剂的新作用。
重要性
人巨细胞病毒（HCMV）的 pTRS1 和 pIRS1 蛋白是 HCMV 复制的关键调节剂，无论是在初次感染期间还是在病毒潜伏性重新激活期间。因此，定义 pTRS1/pIRS1 的分子功能对于理解控制 HCMV 复制和病毒疾病的分子事件非常重要。这些数据为 pTRS1 功能作用提供了新的见解，为其他人理解感染细胞生物学的新特征提供了起点。这项研究的另一个重要结果是发现特定的蛋白磷酸酶 1（PP1）调节亚基是抑制 PKR/eIF2α 信号所必需的，这是一种对抗病毒感染的重要细胞先天免疫防御。这些数据为未来发现破坏 pTRS1 与 PP1 相互作用的治疗方法奠定了基础，从而使细胞防御能够限制 HCMV 复制和疾病。
