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

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.
Author Summary After infecting a target cell, HIV-1 like all retroviruses converts the viral single strand RNA genome into double strand DNA by the process called reverse transcription. Host proteins are known to be important for reverse transcription yet a direct role for any host protein has not been demonstrated. In this paper, we show that a eukaryotic translation elongation factor (eEF1A), an abundant cellular protein, directly and strongly binds to the viral enzyme reverse transcriptase (RT). The biological relevance of the association is supported by mutational analysis of RT and by treating cells with the small molecule didemnin B that specifically binds eEF1A. Mutation of RT or treatment of cells with didemnin B resulted in significantly decreased efficiency of reverse transcription. Didemnin B treatment of cells disrupted HIV-1’s ability to maintain the viral machinery necessary for reverse transcription. However, an HIV-1 mutant, which does not interact with eEF1A, was resistant to didemnin B negative effects on early viral replication, showing that didemnin B affects HIV-1 by targeting the RT-eEF1A interaction. Altogether, this study demonstrates that eEF1A is an integral component of the viral reverse transcription complex and that the RT-eEF1A interaction is a possible new drug target to inhibit HIV-1 replication.