A CONSERVED DOMAIN AND MEMBRANE TARGETING OF NEF FROM HIV AND SIV AREREQUIRED FOR ASSOCIATION WITH A CELLULAR SERINE KINASE-ACTIVITY

Among the primate lentiviruses (human immunodeficiency virus (HPV) -1, HIV-2, and simian immunodeficiency virus (SIV)), the nef gene is high ly conserved and encodes a myristylated protein of similar to 27 kDa ( HIV-1) or similar to 34 kDa (HIV-2, SIV). Previously, we found Nef exp ressed either as a CDS-Nef fusion protein or as a native protein in vi rally infected T cell lines associates with a cellular serine kinase. This kinase activity phosphorylated two proteins of 62 and 72 kDa that coimmunoprecipitate with Nef in in vitro kinase assays. Using transie nt expression, various Nef alleles and mutants have been analyzed for association with the cellular kinase activity. The ability of Nef to a ssociate with the kinase activity is conserved among several alleles o f HIV-1 as well as SIVmac239 and is observed in non-lymphoid cell line s of simian and murine origins. Two separate regions of HIV-1(SF2) Nef are critical for the associated kinase activity. One domain overlaps with a central highly conserved region found in all primate lentivirus nef genes and has been provisionally mapped to amino acids 45-127. Be cause membrane localization of Nef is important for the associated cel lular kinase activity, the second domain represents a membrane targeti ng signal. Moreover, point mutations within the central region that ab rogate the Nef-associated kinase activity in HIV-1(SF2) Nef have the s ame effect when introduced into SIVmac239open Nef.