Comprehensive Analyses of Intraviral Epstein-Barr Virus Protein-Protein Interactions Hint Central Role of BLRF2 in the Tegument Network
Comprehensive Analyses of Intraviral Epstein-Barr Virus Protein-Protein Interactions Hint Central Role of BLRF2 in the Tegument Network
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DOI：
10.1128/jvi.00518-22
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发表时间：
2022-07-11
影响因子：
5.4
通讯作者：
Murata, Takayuki
中科院分区：
文献类型：
--
作者：
Hara, Yuya;Watanabe, Takahiro;Murata, Takayuki
EBV仍然是一个重大的公共卫生挑战，引起传染性单核细胞增多症和几种癌症类型。因此，更好地了解eb病毒复制的分子机制具有重要的临床意义。蛋白质-蛋白质相互作用（PPIs）对各种生物过程至关重要。eb病毒（EBV）蛋白通常形成复合物，调节人类细胞中病毒基因组的复制和持久性。然而，EBV蛋白复合物在生理条件下的作用尚不清楚。在这项研究中，我们使用NanoBiT系统对活细胞中的EBV PPIs进行了全面分析。我们确定了195例ppi，其中许多以前没有报道过。这些PPIs的计算分析显示，仅在γ疱疹病毒中发现的BLRF2是EBV被膜蛋白结构网络中的中心蛋白。为了表征BLRF2的作用，我们使用CRISPR/Cas9生成了两个BLRF2敲除EBV克隆。BLRF2敲除显著降低了感染性病毒颗粒的产生，外源表达BLRF2可部分恢复感染性病毒颗粒的产生。此外，还发现了BLRF2蛋白的自结合，而自结合关键残基的突变影响了该蛋白的稳定性。我们的数据表明，BLRF2是一个在子代病毒粒子成熟过程中起重要作用的被膜网络枢纽。EBV仍然是一个重大的公共卫生挑战，引起传染性单核细胞增多症和几种癌症类型。因此，更好地了解eb病毒复制的分子机制具有重要的临床意义。由于蛋白-蛋白相互作用（PPIs）是病毒相关发病机制的主要调节因子，因此对PPIs进行全面分析是必要的。先前对EBV或其他疱疹病毒中PPIs的研究主要采用酵母双杂交（Y2H）系统、免疫沉淀和拉下试验。在此，我们使用一种新颖的基于发光的方法，鉴定了195个ppi，其中大多数以前未被报道过。利用敲除病毒进行的计算和功能分析显示，BLRF2在EBV生命周期中起着核心作用，这使其成为药物开发的一个有价值的靶点。
EBV remains a significant public health challenge, causing infectious mononucleosis and several cancer types. Therefore, the better understanding of the molecular mechanisms underlying EBV replication is of high clinical importance.Protein-protein interactions (PPIs) are crucial for various biological processes. Epstein-Barr virus (EBV) proteins typically form complexes, regulating the replication and persistence of the viral genome in human cells. However, the role of EBV protein complexes under physiological conditions remains unclear. In this study, we performed comprehensive analyses of EBV PPIs in living cells using the NanoBiT system. We identified 195 PPIs, many of which have not previously been reported. Computational analyses of these PPIs revealed that BLRF2, which is only found in gammaherpesviruses, is a central protein in the structural network of EBV tegument proteins. To characterize the role of BLRF2, we generated two BLRF2 knockout EBV clones using CRISPR/Cas9. BLRF2 knockout significantly decreased the production of infectious virus particles, which was partially restored by exogenous BLRF2 expression. In addition, self-association of BLRF2 protein was found, and mutation of the residues crucial for the self-association affected stability of the protein. Our data imply that BLRF2 is a tegument network hub that plays important roles in progeny virion maturation. IMPORTANCE EBV remains a significant public health challenge, causing infectious mononucleosis and several cancer types. Therefore, the better understanding of the molecular mechanisms underlying EBV replication is of high clinical importance. As protein-protein interactions (PPIs) are major regulators of virus-associated pathogenesis, comprehensive analyses of PPIs are essential. Previous studies on PPIs in EBV or other herpesviruses have predominantly employed the yeast two-hybrid (Y2H) system, immunoprecipitation, and pulldown assays. Herein, using a novel luminescence-based method, we identified 195 PPIs, most of which have not previously been reported. Computational and functional analyses using knockout viruses revealed that BLRF2 plays a central role in the EBV life cycle, which makes it a valuable target for drug development.