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. Host cell factors are essential for these processes, although very few reverse transcription complex (RTC) binding host cell factors have been convincingly shown to affect uncoating or reverse transcription. We previously reported that cellular eukaryotic elongation factor 1A (eEF1A) interacts tightly and directly with HIV-1 reverse transcriptase (RT), which is important for HIV-1 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 the surface exposed acidic thumb domain residues D250, E297, E298 and E300 to positively charged arginine residues resulted in various levels of impairment on RT and eEF1A interaction. This indicates that surface electrostatics play a critical role in the interaction between this negatively charged region in the RT thumb domain and the positively charged eEF1A protein. The impairment of RT and eEF1A interaction by the RT mutations correlated to the efficiencies of reverse transcription, uncoating and infectivity. The best example of this is the strictly conserved E300 residue, where mutation significantly impaired the RT interaction with eEF1A without affecting in vitro RT catalytic activity, and strongly inhibited virus replication in CD4+ T cells. This study advances the structural and mechanistic detail about the key RT:eEF1A interaction in HIV-1 infection and implicates its importance in uncoating for the first time. This provides further basis for development of a RT:eEF1A interaction inhibiting antiviral drug for HIV-1, and suggests the surface exposed acidic patch of the RT thumb domain may be an attractive drug target. Preliminary RT:eEF1A interaction inhibitors identified through a drug screen will be discussed.