Abstract
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.
Highlights
To convert the viral genomic RNA into double stranded DNA, HIV-1 forms a prototypical ribonucleoprotein complex on the viral genomic RNA called the reverse transcription complex (RTC) [1]
We show that a eukaryotic translation elongation factor, an abundant cellular protein, directly and strongly binds to the viral enzyme reverse transcriptase (RT)
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
Summary
To convert the viral genomic RNA into double stranded DNA, HIV-1 forms a prototypical ribonucleoprotein complex on the viral genomic RNA called the reverse transcription complex (RTC) [1]. It includes the viral enzymes reverse transcriptase (RT) and integrase (IN), as well as other viral proteins including capsid (CA), matrix (MA), viral protein R (Vpr), and viral infectivity factor (Vif). HIV-1 reverse transcription begins shortly after the viral core enters the cell cytoplasm, when nucleotides become available to make a short single strand of DNA referred to as minus strand strong stop DNA (sssDNA or early DNA). Studies show that completion of in vitro reverse transcription can be greatly enhanced by the addition of cell lysate, indicating that one or more cellular activities are required to form RTC and enable its full function [6,7,8]
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