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 and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P-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, but not that of tRNA, 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 showed greater inhibition of MA membrane binding than full-length tRNA. While transplantation of the D loop sequence of tRNA into tRNA resulted in a modest increase in the inhibitory effect relative to WT tRNA, replacing the entire D arm sequence with that of tRNA was necessary to confer the full inhibitory effects upon tRNA. Together, these results demonstrate that the D arm of tRNA 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.