Mutations that mimic phosphorylation of the HIV-1 matrix protein do not perturb the myristyl switch.
Recent studies indicate that the matrix domain (MA) of the HIV-1 Gag polyprotein directs Gag to the plasma membrane for virus assembly via a phosphatidylinositol-4,5-bisphosphate (PIP(2))-dependent myristyl switch mechanism. MA also has been reported to direct nuclear trafficking via nuclear import and export functions, and some studies suggest that nuclear targeting may be regulated by MA phosphorylation (although this proposal remains controversial). We have prepared and studied a series of HIV-1 MA mutants containing Ser-to-Asp substitutions designed to mimic phosphorylation, including substitutions in regions of the protein involved in protein-protein interactions and known to influence the myristyl switch (S6D, S9D, S67D, S72D, S6D/S9D, and S67D/S72D). We were particularly interested in substitutions at residue 6, since conservative mutations adjacent to this site strongly perturb the myristyl switch equilibrium, and this site had not been genetically tested due to its involvement in post-translational myristylation. Our studies reveal that none of these mutations, including S6D, influences the PIP(2)- or concentration-dependent myristyl switch equilibrium. In addition, all of the mutants bind liposomes with affinities that are only slightly reduced in comparison with the native protein. In contrast, the myristylated mutants bind liposomes with substantially greater affinity than that of the native, unmyristylated protein. These findings support the hypothesis that phosphorylation is unlikely to significantly influence membrane-mediated intracellular trafficking.
Saad
Jamil S
JS
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, Maryland 21250, USA.
Kim
Andrew
A
Ghanam
Ruba H
RH
Dalton
Amanda K
AK
Vogt
Volker M
VM
Wu
Zhibin
Z
Lu
Wuyuan
W
Summers
Michael F
MF
D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics; D019814:Myristic Acid / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D017354:Point Mutation; D012694:Serine / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D015683:Gene Products, gag; D019814:Myristic Acid; D001224:Aspartic Acid; D012694:Serine