An integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus.
An integrated approach to elucidate the intra-viral and viral-cellular protein interaction networks of a gamma-herpesvirus.
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DOI：
10.1371/journal.ppat.1002297
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发表时间：
2011-10
期刊：
影响因子：
6.7
通讯作者：
Sun R
中科院分区：
文献类型：
--
作者：
Lee S;Salwinski L;Zhang C;Chu D;Sampankanpanich C;Reyes NA;Vangeloff A;Xing F;Li X;Wu TT;Sahasrabudhe S;Deng H;Lacount DJ;Sun R
通过全基因组酵母双杂交(Y2H)筛选，对小鼠疱疹病毒68(γ-68)编码的开放阅读框(ORF)的分子功能进行了研究。构建了84个MHV-68基因和基因片段的文库，并将其转入Y2H载体。在Y2H试验中测试了病毒蛋白之间所有可能的成对相互作用，从而确定了23种病毒内蛋白质-蛋白质相互作用(PPI)。免疫共沉淀实验证实了病毒蛋白间70%的相互作用。为了系统地研究病毒与细胞蛋白的相互作用，根据细胞cDNA文库筛选MHV-68 Y2H结构，产生243个病毒与细胞蛋白相互作用的PPI，涉及197种不同的细胞蛋白。网络分析表明，MHV-68靶向的细胞蛋白在细胞PPI网络中有更多的伙伴，并且彼此之间的位置比预期的更近。利用这一观察结果，我们根据细胞蛋白与其他MHV-68相互作用蛋白的网络距离对它们进行了评分，并将它们分为高优先级(Y2H-HP)和低优先级/未评分(Y2H-LP/NS)组。当siRNAs抑制MHV-68的表达时，来自Y2H-HP的基因显著地改变了MHV-68的复制(来自Y2H-HP的基因的53%，来自Y2H-LP/NS的基因的21%，以及从人类PPI网络中随机选择的16%的基因；p<；0.05)。Y2H-HP组丰富的基因本体论(GO)术语包括细胞凋亡调控、蛋白激酶级联、翻译后蛋白修饰、核糖核酸聚合酶II启动子转录、IκB激酶/核因子κB级联。功能验证分析表明，与MHV-68 ORF34相互作用的PCBP1可能以与其病毒相互作用伙伴的作用相一致的方式参与调控晚期病毒基因的表达。我们的研究将Y2H筛查与多种功能验证方法相结合，以创建γ-疱疹病毒-病毒和病毒-细胞蛋白相互作用网络。疱疹病毒Epstein Barr病毒(EBV)和卡波西肉瘤疱疹病毒(KSHV)的持续感染与肿瘤的形成有关。为了更好地了解这些病毒和其他相关病毒是如何与宿主细胞相互作用以促进病毒复制和导致疾病的，我们研究了小鼠伽玛-疱疹病毒68(MHV-68)。MHV-68与EBV和KSHV属于同一组疱疹病毒，但具有在细胞培养中能够高效复制的优势。我们的研究使用全基因组筛选来鉴定80种MHV-68蛋白之间的23种蛋白质-蛋白质相互作用。这些相互作用中的几个可能对组装新病毒很重要。我们还发现了MHV-68与细胞蛋白之间的243个相互作用。为了帮助确定后续研究中细胞蛋白质的优先顺序，我们开发了一种新的计算工具来分析我们的数据。优先级分数高的蛋白质比优先级低的蛋白质更有可能影响病毒复制。对MHV-68复制影响最大的细胞蛋白是PCBP1，它负向调节MHV-68晚期基因的表达。这项研究发现了许多与MHV-68复制有关的新的细胞蛋白质，并建立了一种从高通量病毒-细胞蛋白质-蛋白质相互作用数据集中识别重要蛋白质的方法。
Genome-wide yeast two-hybrid (Y2H) screens were conducted to elucidate the molecular functions of open reading frames (ORFs) encoded by murine γ-herpesvirus 68 (MHV-68). A library of 84 MHV-68 genes and gene fragments was generated in a Gateway entry plasmid and transferred to Y2H vectors. All possible pair-wise interactions between viral proteins were tested in the Y2H assay, resulting in the identification of 23 intra-viral protein-protein interactions (PPIs). Seventy percent of the interactions between viral proteins were confirmed by co-immunoprecipitation experiments. To systematically investigate virus-cellular protein interactions, the MHV-68 Y2H constructs were screened against a cellular cDNA library, yielding 243 viral-cellular PPIs involving 197 distinct cellar proteins. Network analyses indicated that cellular proteins targeted by MHV-68 had more partners in the cellular PPI network and were located closer to each other than expected by chance. Taking advantage of this observation, we scored the cellular proteins based on their network distances from other MHV-68-interacting proteins and segregated them into high (Y2H-HP) and low priority/not-scored (Y2H-LP/NS) groups. Significantly more genes from Y2H-HP altered MHV-68 replication when their expression was inhibited with siRNAs (53% of genes from Y2H-HP, 21% of genes from Y2H-LP/NS, and 16% of genes randomly chosen from the human PPI network; p<0.05). Enriched Gene Ontology (GO) terms in the Y2H-HP group included regulation of apoptosis, protein kinase cascade, post-translational protein modification, transcription from RNA polymerase II promoter, and IκB kinase/NFκB cascade. Functional validation assays indicated that PCBP1, which interacted with MHV-68 ORF34, may be involved in regulating late virus gene expression in a manner consistent with the effects of its viral interacting partner. Our study integrated Y2H screening with multiple functional validation approaches to create γ-herpes viral-viral and viral-cellular protein interaction networks. Persistent infections by the herpesviruses Epstein Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are associated with tumor formation. To better understand how these and other related viruses interact with their host cells to promote virus replication and cause disease, we studied murine gamma-herpesvirus 68 (MHV-68). MHV-68 belongs to the same group of herpesviruses as EBV and KSHV, but has the advantage of being able to replicate efficiently in cell culture. Our study used genome-wide screens to identify 23 protein-protein interactions between the 80 MHV-68 proteins. Several of these interactions are likely to be important for assembling new viruses. We also discovered 243 interactions between MHV-68 and cellular proteins. To help prioritize cellular proteins for follow up studies, we developed a new computational tool to analyze our data. Proteins with high priority scores were more likely to affect viral replication than low priority proteins. Among the cellular proteins that had the greatest effect on MHV-68 replication was PCBP1, which negatively regulated MHV-68 late gene expression. This study identified many novel cellular proteins involved in MHV-68 replication and established a method to identify important proteins from high-throughput virus-cellular protein-protein interaction data sets.
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影响因子：
5.4
作者：
Arumugaswami, Vaithilingaraja;Wu, Ting-Ting;Sun, Ren
通讯作者：
Sun, Ren
影响因子：
5.4
作者：
Bortz, Eric;Wang, Lili;Sun, Ren
通讯作者：
Sun, Ren
影响因子：
5.4
作者：
Arumugaswami, Vaithilingaraja;Sitapara, Ronika;Sun, Ren
通讯作者：
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影响因子：
14.9
作者：
Chatr-aryamontri, Andrew;Ceol, Arnaud;Palazzi, Luisa Montecchi;Nardelli, Giuliano;Schneider, Maria Victoria;Castagnoli, Luisa;Cesareni, Gianni
通讯作者：
Cesareni, Gianni
影响因子：
6.7
作者：
Fossum E;Friedel CC;Rajagopala SV;Titz B;Baiker A;Schmidt T;Kraus T;Stellberger T;Rutenberg C;Suthram S;Bandyopadhyay S;Rose D;von Brunn A;Uhlmann M;Zeretzke C;Dong YA;Boulet H;Koegl M;Bailer SM;Koszinowski U;Ideker T;Uetz P;Zimmer R;Haas J
通讯作者：
Haas J