HIV-1 MATRIX INTERACTIONS WITH tRNAS AND THEIR ROLE IN MEMBRANE TARGETING
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Date
2017-01-01
Type of Work
Department
Chemistry & Biochemistry
Program
Biochemistry
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Access limited to the UMBC community. Item may possibly be obtained via Interlibrary Loan through a local library, pending author/copyright holder's permission.
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Abstract
Assembly of the HIV-1 virion begins when the myristoylated matrix domain (myrMA) of the Gag polyprotein targets the Gag-genome complex to the plasma membrane through interactions between myrMA'shighly basic region (HBR) and the membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In addition to interacting with PI(4,5)P2, myrMA will bind to specific tRNAs in the cytoplasm, and studies have found that treating myrMA with RNase will decrease myrMA'sability to discriminate between membranes containing and lacking PI(4,5)P2. Here we developed assays to characterize the interaction between MA and tRNALys3, one of the RNAs that bound to MA and the primer used during reverse transcription. NMR spectrometry mapped the RNA-binding region to the HBR, including lysine residues known to regulate membrane binding. Isothermal titration calorimetry (ITC) of basic patch mutants determined that many mutations known to affect plasma membrane binding also decreased myrMA'saffinity to tRNALys3. ITC experiments also found that exposure of MA'sN-terminal myristoyl group weakens tRNA-MA interactions, supporting the hypothesis that tRNA binding regulates membrane binding as myristoyl exposure occurs upon binding to the plasma membrane. However, 1H-1D NMR liposome competition assays determined that the presence of tRNA alone is not enough to allow myrMA to discriminate between membranes. tRNALys3 prevented binding to all membranes, including those containing high levels of PI(4,5)P2 and those with raft-like compositions. Lowering the pH of the samples to induce myristoyl exposure did induce specific membrane binding in the presence of tRNA, suggesting some other mechanism is required in addition to tRNA interactions to regulate assembly on the plasma membrane.