Abstract
ABSTRACTOnce HIV-1 enters a cell, the viral core is uncoated by a poorly understood mechanism and the HIV-1 genomic RNA is reverse transcribed into DNA. Host cell factors are essential for these processes, although very few reverse transcription complex binding host cell factors have been convincingly shown to affect uncoating or reverse transcription. We previously reported that cellular eukaryotic translation elongation factor 1A (eEF1A) interacts tightly and directly with HIV-1 reverse transcriptase (RT) for more efficient reverse transcription. Here we report that the surface-exposed acidic residues in the HIV-1 RT thumb domain alpha-J helix and flanking regions are important for interaction with eEF1A. Mutation of surface-exposed acidic thumb domain residues D250, E297, E298, and E300 to arginine resulted in various levels of impairment of the interaction between RT and eEF1A. This indicates that this negatively charged region in the RT thumb domain is important for interaction with the positively charged eEF1A protein. The impairment of RT and eEF1A interaction by the RT mutations correlated with the efficiency of reverse transcription, uncoating, and infectivity. The best example of this is the strictly conserved E300 residue, where mutation significantly impaired the interaction of RT with eEF1A and virus replication in CD4+ T cells without affecting in vitro RT catalytic activity, RT heterodimerization, or RNase H activity. This study demonstrated that the interaction between surface-exposed acidic residues of the RT thumb domain and eEF1A is important for HIV-1 uncoating, reverse transcription, and replication.
Highlights
Once HIV-1 enters a cell, the viral core is uncoated by a poorly understood mechanism and the HIV-1 genomic RNA is reverse transcribed into DNA
E297, E298, and E300 are a highly surface-exposed (Fig. S1) cluster of acidic residues in the alpha-J helix of the reverse transcriptase (RT) thumb domain and do not contact other RT domains in the crystal structure (Fig. 2A). To investigate if these residues are important for interaction with eukaryotic translation elongation factor 1A (eEF1A), the D250, E297, E298, and E300 residues were mutated to alanine (A) and arginine (R)
Previously, we demonstrated that eEF1A binds tightly and directly to HIV-1 RT and that this is important for reverse transcription efficiency [18]
Summary
Once HIV-1 enters a cell, the viral core is uncoated by a poorly understood mechanism and the HIV-1 genomic RNA is reverse transcribed into DNA. This study demonstrated that the interaction between surface-exposed acidic residues of the RT thumb domain and eEF1A is important for HIV-1 uncoating, reverse transcription, and replication. The trafficking of the HIV-1 core and reverse transcription complex (RTC) from the cell periphery to the nucleus is tightly regulated by viral and host proteins [2], but the process is poorly understood. Identifying how specific host cell factors facilitate optimal uncoating and reverse transcription is important to better understand these early stages of HIV-1 replication. This could lead to the development of a different class of drugs that target virus-host interactions required for early stages of HIV-1 replication [15, 16]. Mutation of these residues to alanine was predicted to severely destabilize the RT protein structure [18] and possibly disrupt the RT region that directly contacts eEF1A, but this region has yet to be identified
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