ALTERED PLAQUE-FORMATION BY RECOMBINANT VACCINIA VIRUS EXPRESSING SIMIAN IMMUNODEFICIENCY VIRUS NEF

The nef gene of primate lentiviruses encodes a myristoylated protein t hat is important for pathogenicity and the maintenance of high virus l oads. A deletion in nef leads to a significant reduction of the pathog enicity of simian immunodeficiency virus (SIV) in macaques. At the cel lular and biochemical levels, Nef has been shown to down-regulate CD4 and major histocompatibility complex class I molecules and to interact with cellular protein kinases. The importance of these activities for Nef function remains uncertain. We have prepared vaccinia virus recom binants expressing different alleles of SIV nef. When grown on TK(-)13 3 cells, recombinants constructed with the nef allele from SIVmac1A11 produced typical plaques while recombinants expressing the nef allele from SIVmac239-R1 gave rise to plaques with altered morphology. By usi ng chimeric Nef proteins and site-directed mutagenesis, the amino acid responsible for altered plaque formation was mapped to a leucine at r esidue 211. In vitro phosphorylation of immunoprecipitates prepared fr om cells infected with the vaccinia virus recombinants resulted in lab eled proteins of 62 and 90 kDa. The recombinants differed in the abili ty to stimulate phosphorylation, and the leucine at residue 211 was ag ain found to be the determining amino acid. These results might help e lucidate the role of nef in the pathogenesis of SIV.