HIV-1 Matrix Protein Interactions with tRNA: Implications for Membrane Targeting |
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Authors: | Christy R. Gaines Emre Tkacik Amalia Rivera-Oven Phoebe Somani Alecia Achimovich Tawakalitou Alabi Angela Zhu Noel Getachew Ae Lim Yang Matthew McDonough Tarik Hawkins Zoe Spadaro Michael F. Summers |
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Affiliation: | Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250, USA |
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Abstract: | The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNALys3), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNALys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNALys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. Unexpectedly, the affinity of MA for tRNALys3 (Kd = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P2-enriched liposomes (Kd(apparent) = 10.2 ± 2.1 μM), and NMR studies indicate that tRNALys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P2, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNALys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag–Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled. |
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Keywords: | HIV matrix protein tRNA membrane liposomes PM plasma membrane MA myristoylated matrix NC nucleocapsid 2 phosphatidylinositol-4,5-bisphosphate PS phosphatidylserine ITC isothermal titration calorimetry EMSAs electrophoretic mobility shift assays |
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