Li Dongsheng, Wei Ting, Rawle Daniel J, Qin Fangyun, Wang Rui, Soares Dinesh C, Jin Hongping, Sivakumaran Haran, Lin Min-Hsuan, Spann Kirsten, Abbott Catherine M, Harrich David
Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.
School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, Australia.
PLoS Pathog. 2015 Dec 1;11(12):e1005289. doi: 10.1371/journal.ppat.1005289. eCollection 2015 Dec.
Reverse transcription is the central defining feature of HIV-1 replication. We previously reported that the cellular eukaryotic elongation factor 1 (eEF1) complex associates with the HIV-1 reverse transcription complex (RTC) and the association is important for late steps of reverse transcription. Here we show that association between the eEF1 and RTC complexes occurs by a strong and direct interaction between the subunit eEF1A and reverse transcriptase (RT). Using biolayer interferometry and co-immunoprecipitation (co-IP) assays, we show that association between the eEF1 and RTC complexes occurs by a strong (KD ~3-4 nM) and direct interaction between eEF1A and reverse transcriptase (RT). Biolayer interferometry analysis of cell lysates with titrated levels of eEF1A indicates it is a predominant cellular RT binding protein. Both the RT thumb and connection domains are required for interaction with eEF1A. A single amino acid mutation, W252A, within the thumb domain impaired co-IP between eEF1A and RT, and also significantly reduced the efficiency of late reverse transcription and virus replication when incorporated into infectious HIV-1. Molecular modeling analysis indicated that interaction between W252 and L303 are important for RT structure, and their mutation to alanine did not impair heterodimerisation, but negatively impacted interaction with eEF1A. Didemnin B, which specifically binds eEF1A, potently inhibited HIV-1 reverse transcription by greater than 2 logs at subnanomolar concentrations, especially affecting reverse transcription late DNA synthesis. Analysis showed reduced levels of RTCs from HIV-1-infected HEK293T treated with didemnin B compared to untreated cells. Interestingly, HIV-1 with a W252A RT mutation was resistant to didemnin B negative effects showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. The combined evidence indicates a direct interaction between eEF1A and RT is crucial for HIV reverse transcription and replication, and the RT-eEF1A interaction is a potential drug target.
逆转录是HIV-1复制的核心决定性特征。我们之前报道过，细胞真核延伸因子1（eEF1）复合物与HIV-1逆转录复合物（RTC）相关联，且这种关联对逆转录的后期步骤很重要。在此我们表明，eEF1与RTC复合物之间的关联是通过亚基eEF1A与逆转录酶（RT）之间强烈而直接的相互作用发生的。使用生物层干涉术和免疫共沉淀（co-IP）分析，我们表明eEF1与RTC复合物之间的关联是通过eEF1A与逆转录酶（RT）之间强烈的（解离常数KD约为3 - 4 nM）直接相互作用发生的。对含有滴定水平eEF1A的细胞裂解物进行生物层干涉术分析表明，它是主要的细胞RT结合蛋白。RT的拇指结构域和连接结构域对于与eEF1A相互作用都是必需的。拇指结构域内的单个氨基酸突变W252A损害了eEF1A与RT之间的免疫共沉淀，并且当整合到有传染性的HIV-1中时，也显著降低了后期逆转录和病毒复制的效率。分子建模分析表明，W252与L303之间的相互作用对RT结构很重要，将它们突变为丙氨酸并不损害异二聚化，但对与eEF1A的相互作用有负面影响。特异性结合eEF1A的Didemnin B在亚纳摩尔浓度下能有效抑制HIV-1逆转录超过2个对数级，尤其影响逆转录后期的DNA合成。分析表明，与未处理的细胞相比，用Didemnin B处理的HIV-1感染的HEK293T细胞中RTC的水平降低。有趣的是，具有W252A RT突变的HIV-1对Didemnin B的负面影响具有抗性，这表明Didemnin B通过靶向RT - eEF1A相互作用来影响HIV-1。综合证据表明，eEF1A与RT之间的直接相互作用对HIV逆转录和复制至关重要，且RT - eEF1A相互作用是一个潜在的药物靶点。
