The acidic region and conserved putative protein kinase C phosphorylation site in Nef are important for SIV replication in rhesus macaques

Variants of the pathogenic SIVmac239 clone with changes in Nef were analyze d to assess the functional relevance of two highly conserved regions in Nef in vitro and in vivo. Changes in a region with an acidic charge (Aci-Nef), or a potential protein kinase C phosphorylation site (PKC-Nef), impaired t he ability of Nef to down-regulate CD4 and MHC class I surface expression a nd to alter CD3-initiated signal transduction in Jurkat T cells. The Aci-Ne f, but not the PKC-Nef, associated with the previously described p65 phosph oprotein. SIV containing Aci-Nef, but not SIV containing PKC-Nef, showed re duced infectivity and replication in cell culture systems. One of two rhesu s macaques infected with the PKC-Nef mutant virus showed rapid reversion an d progressed to disease. In the second animal no reversions and nonprogress ive infection was observed. In one of two macaques infected with the Aci-Ne f variant, the mutations were stable during the first 40 weeks after infect ion. Thereafter, variants evolved in which up to six of the eight mutated p ositions in Nef were reverted and functional activity in vitro was partiall y restored. These changes occurred concomitantly with increasing viral load and disease progression. The second animal infected with the Aci-Nef varia nt showed no reversions and remained asymptomatic. Our study suggests that the acidic region and conserved PKC phosphorylation site in Nef are importa nt for SIV replication in rhesus macaques and for several in vitro Nef func tions. An almost wild-type activity in in vitro infectivity and replication assays seems insufficient to confer a full nef-positive phenotype in vivo. (C) 1999 Academic Press.