THE MAJOR HOMOLOGY REGION OF THE HIV-1 GAG PRECURSOR INFLUENCES MEMBRANE AFFINITY

Assembly of retroviruses, including HIV-1, involves movement of newly synthesized viral proteins and RNA to the plasma membranes of host cel ls. The major homology region (MHR, aa 285-304), a highly conserved se quence in the capsid domain of the HIV-1 Gag precursor polyprotein, pl ays a critical, but unknown, role in infectious particle assembly. Mut ations of invariant residues in the sequence have pleiotropic effects: Mutation of Gln287 blocks viral assembly while mutation of Arg299 per mits assembly, but blocks formation of infectious particles. In this r eport, we demonstrate that Gag proteins lacking the entire MHR accumul ated in the cytoplasm of transfected COS-1 cells, as did the wild-type protein, but were processed in a defective manner at the cellular mem brane resulting in impaired particle assembly. To further examine the role of the MHR in membrane association, membrane binding of unmyristy lated recombinant Gag proteins with alterations in the MHR was investi gated in vitro. The wild-type Gag precursor bound to acidic phospholip id vesicles highly efficiently, as determined by fluorescence spectros copy or velocity sedimentation. In contrast, deletion of the entire MH R reduced membrane affinity an average of similar to 3-fold or greater . Mutation of the invariant Gln287 residue disrupted membrane affinity similar to 6-fold relative to the wild-type, which was similar to the level of inhibition obtained by deletion of a membrane-binding signal previously identified in the matrix domain of the Gag precursor. Muta tion of the invariant Arg299 residue reduced the affinity to a lesser extent. The results indicate that correct membrane binding is determin ed not only by signals in the MA domain of the precursor but also by s equences in the CA domain of Gag. We speculate that defects in the hig hly conserved MHR affect a Gag conformation that is required for produ ctive interactions at the membrane assembly site.