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

Overview

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)P₂ and tRNA-mediated inhibition of non-specific or premature membrane binding. Different tRNAs inhibit PI(4,5)P₂-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 full-length tRNA^Pro. While transplantation of the D loop sequence of tRNA^Lys3 into tRNA^Pro 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 tRNA^Pro. 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.