Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.
Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.
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DOI：
10.1371/journal.ppat.1011682
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发表时间：
2023-10
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
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人巨细胞病毒（HCMV）编码多种假定的G蛋白偶联受体（GPCR）。US 28作为病毒趋化因子受体发挥作用，并在病毒感染的潜伏期和裂解期表达。US 28积极促进细胞迁移、转化，并在介导病毒潜伏期和再活化中发挥重要作用;然而，关于这些过程中涉及的相互作用伙伴的知识仍然不完整。在此，我们利用邻近依赖性生物素化酶（TurboID）来表征US 28相互作用组在分离表达时以及在潜伏性（CD 34+造血祖细胞）和裂解性（成纤维细胞）HCMV感染期间的特征。我们的分析表明，US 28信号体与RhoA和EGFR信号转导通路会聚，共享多种介质，这些介质是细胞增殖和分化等过程中的主要参与者。通过免疫印迹和小分子抑制剂在功能测定中验证了US 28信号传导复合物的组成成员。重要的是，我们将RhoGEFs确定为关键的US 28信号中间体。利用用小分子抑制剂Rhosin或Y16处理的原代CD 34+造血祖细胞（HPC）的体外潜伏期和再活化测定表明，US 28-RhoGEF相互作用是有效的病毒再活化所必需的。使用人源化小鼠模型在体内重现了这些发现，其中RhoGEF的抑制导致病毒重新激活失败。总之，我们的数据确定了US 28相互作用组中的多种新蛋白质，这些蛋白质在病毒潜伏期和再活化中发挥重要作用，突出了接近传感器标记在表征蛋白质相互作用组中的作用，并为开发新型抗HCMV治疗药物提供了深入了解。人巨细胞病毒（HCMV）仍然是世界范围内最流行的病毒感染之一。HCMV的原发性感染通常是无症状的，并导致在髓系细胞内建立潜伏期。一旦潜伏期建立，病毒将在宿主的整个生命周期中持续存在。随后的病毒再激活事件可对免疫功能低下的人群（包括移植受者和艾滋病患者）造成危及生命的健康并发症。许多因素已被证明介导从潜伏性到裂解性HCMV感染的转换，例如通过病毒G蛋白偶联受体（vGPCR）的信号转导US 28。在本报告中，我们利用邻近依赖性标记结合质谱法，以确定主机和病毒蛋白接近US 28。我们的分析表明US 28与EGFR和RhoA信号通路之间存在显著重叠。我们进一步探讨了US 28和RhoA信号转导通路之间的关系，以确定RhoGEFs作为US 28信号体的重要成员。我们的数据表明，消融RhoGEF活性显着减弱US 28信号。此外，我们表明，RhoGEFs的药理学抑制导致病毒无法在体外和体内有效地重新激活。这些发现揭示了以前未知的US 28相互作用因子，其在促进病毒再活化中起着不可或缺的作用，并提供了与US 28功能和体内病毒再活化有关的特异性细胞因子的第一个例子。
Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 –RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics. Human cytomegalovirus (HCMV), continues to be amongst the most prevalent viral infections worldwide. Primary infection of HCMV is often asymptomatic and results in the establishment of latency within cells of myeloid lineage. Once latency is established, the virus will persist throughout the host’s lifetime. Subsequent viral reactivation events can pose life-threatening health complications for the immunocompromised population; including transplant recipients and AIDS patients. Many factors have been shown to mediate the switch from latent to lytic HCMV infection such as signal transduction through the viral G protein-coupled receptor (vGPCR) US28. In the present report, we utilize proximity-dependent labeling coupled with mass spectrometry to identify host and viral proteins proximal to US28. Our analysis indicates significant overlap between US28 and the EGFR and RhoA signaling pathways. We further explored the relationship between US28 and the RhoA signal transduction pathway to identify RhoGEFs as an important member of the US28 signalosome. Our data indicates that ablation of RhoGEF activity significantly attenuates US28 signaling. Furthermore, we show that pharmacological inhibition of RhoGEFs results in an inability of the virus to efficiently reactivate in vitro and in vivo. These findings reveal previously unknown US28 interactors, which play an integral role in the facilitation of viral reactivation, and provide the first example of specific cellular factors being implicated in US28 function and viral reactivation in vivo.
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影响因子：
4.3
作者：
Fabregat A;Korninger F;Viteri G;Sidiropoulos K;Marin-Garcia P;Ping P;Wu G;Stein L;D'Eustachio P;Hermjakob H
通讯作者：
Hermjakob H
影响因子：
4.1
作者：
Daniel Cervantes-Villagrana, Rodolfo;Manuel Color-Aparic, Victor;Vazquez-Prado, Jose
通讯作者：
Vazquez-Prado, Jose
DOI：
10.1073/pnas.0914856107
发表时间：
2010-02-09
影响因子：
11.1
作者：
Aoyagi, Mariko;Gaspar, Miguel;Shenk, Thomas E.
通讯作者：
Shenk, Thomas E.
影响因子：
7.8
作者：
Azoitei, Mihai L.;Noh, Jungsik;Danuser, Gaudenz
通讯作者：
Danuser, Gaudenz
DOI：
10.1006/bbrc.1995.1814
发表时间：
1995-06-06
影响因子：
3.1
作者：
KUHN, DE;BEALL, CJ;KOLATTUKUDY, PE
通讯作者：
KOLATTUKUDY, PE