Specific Interaction between eEF1A and HIV RT Is Critical for HIV-1 Reverse Transcription and a Potential Anti-HIV Target

PLoS Pathogens

Dinesh C. Soares¹  Daniel J. Rawle¹  Ting Wei¹  Haran Sivakumaran¹  Min-Hsuan Lin¹  Dongsheng Li¹  Kirsten Spann¹  Rui Wang¹  Hongping Jin²  David Harrich³  Fangyun Qin⁴  Catherine M. Abbott⁴ 

[1] Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia;Medical Genetics Section, Centre for Genomic and Experimental Medicine, MRC Institute of Genetics and Molecular Medicine, Western General Hospital, University of Edinburgh, Edinburgh, United Kingdom;School of Biomedical Science, Queensland University of Technology, Brisbane, Queensland, Australia;School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, Australia

关键词: HIV-1;    Reverse transcription;    Elongation factors;    Thumbs;    Biosensors;    Small interfering RNAs;    Immunoprecipitation;    Protein interactions;   

DOI  :  10.1371/journal.ppat.1005289

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY