MUTATIONAL ANALYSIS OF THE CONSERVED CYSTEINE RESIDUES IN THE SIMIAN IMMUNODEFICIENCY VIRUS MATRIX PROTEIN

The matrix protein (MA) of human and simian immunodeficiency viruses ( HIV and SIV) is encoded by the amino-terminal region of the Gag precur sor and has been suggested to be involved in different processes durin g the early and late stages of the virus life cycle. The MA protein of SIV contains three cysteine residues at positions 57, 83, and 87, whi ch are also highly conserved among HIV-2 isolates. In order to study t he functional significance of these residues in virus morphogenesis, a series of mutations affecting the cysteines of SIV MA were introduced into a gag-protease construct and expressed in the vaccinia Vector sy stem. The MA mutants were assayed for their ability to synthesize and process the Gag polyprotein precursor as well as to release particles into the culture medium. In addition, the incorporation of the envelop e glycoprotein (Env) into the Gag-made particles was investigated. Sub stitution of alanine for cysteine 87 had little effect on particle rel ease and Env glycoprotein association. By contrast, the individual rep lacement of cysteines 57 or 83 by alanine, as well as the simultaneous mutation of cysteines 83 and 87, significantly reduced the ability of Gag polypeptides to produce extracellular particles. Assembly into pa rticles appeared to be also affected, albeit to a lesser extent, when both cysteines 57 and 83 were replaced by alanine. Furthermore, substi tution of cysteine 83 in the SIV MA domain was found to be detrimental to Gag polyprotein processing. Analysis of the Env glycoprotein assoc iation with recombinant particles revealed that this process was moder ately affected in the case of the double mutants lacking cysteines 57 and 83, or cysteines 57 and 87, and the cysteine-minus triple mutant. Our results suggest that the conserved cysteines 57 and 83 in the MA d omain are important for efficient SIV Gag particle production. (C) 199 5 Academic Press, Inc.