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 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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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