Molecular Determinants in tRNA D-arm Required for Inhibition of HIV-1 Gag Membrane Binding

Plasma-membrane-specific localization of Gag, an essential step in HIV-1 particle assembly, is regulated by the interaction of the Gag MA domain with PI(4,5)P-2 and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P-2-independent membrane binding to varying degrees in vitro; however, the structural determinants for this difference remain unknown. Here we demonstrate that membrane binding of full-length Gag synthesized in vitro using reticulocyte lysates is inhibited when RNAs that contain the anticodon arm of tRNA(Pro), but not that of tRNA(Lys3) , are added exogenously. In contrast, in the context of a liposome binding assay in which the effects of tRNAs on purified MA were tested, full-length tRNA(Lys3) showed greater inhibition of MA membrane binding than fulllength tRNA(Pro). While transplantation of the D loop sequence of tRNA(Lys3) into tRNAPro resulted in a modest increase in the inhibitory effect relative to WT tRNA(Pro), replacing the entire D arm sequence with that of tRNA(Lys3) was necessary to confer the full inhibitory effects upon tRNAPro. Together, these results demonstrate that the D arm of tRNA(Lys3) is a major determinant of strong inhibition of MA membrane binding and that this inhibitory effect requires not only the D loop, which was recently reported to contact the MA highly basic region, but the loop sequence in the context of the D arm structure.(c) 2021 Elsevier Ltd. All rights reserved.