ROLE OF THE SH3-LIGAND DOMAIN OF SIMIAN IMMUNODEFICIENCY VIRUS NEF ININTERACTION WITH NEF-ASSOCIATED KINASE AND SIMIAN AIDS IN RHESUS MACAQUES

The nef gene of the human and simian immunodeficiency viruses (HIV and SIV) is dispensable for viral replication in T-cell lines; however, i t is essential for high virus loads and progression to simian AIDS (SA IDS) in SIV-infected adult rhesus macaques, Nef proteins from HN type 1 (HIV-1), HIV-2, and SIV contain a proline-Xaa-Xaa-proline (PxxP) mot if, The region of Nef with this motif is similar to the Src homology r egion 3 (SH3) ligand domain found in many cell signaling proteins. In virus-infected lymphoid cells, Nef interacts with a cellular serine/th reonine kinase, designated Nef associated kinase (NAK), In this study, analysis of viral clones containing point mutations in the nef gene o f the pathogenic clone SIVmac239 revealed that several strictly conser ved residues in the PxxP region were essential for Nef-NAK interaction . The results of this analysis of Nef mutations in in vitro kinase ass ays indicated that the PxxP region in SIV Nef was strikingly similar t o the consensus sequence for SH3 ligand domains possessing the minus o rientation. To test the significance of the PxxP motif of Nef for vira l pathogenesis, each proline was mutated to an alanine to produce the viral clone SIVmac239-P-104/P(107)A. This clone, expressing Nef that d oes not associate with NAK, was inoculated into seven juvenile rhesus macaques, In vitro kinase assays were performed on virus recovered fro m each animal; the ability of Nef to associate,vith NAK was restored i n five of these animals as early as 8 weeks after infection. Analysis of nef genes from these viruses revealed patterns of genotypic reversi on in the mutated PxxP motif, These revertant genotypes, which include d a second-site suppressor mutation, restored the ability of Nef to in teract with NAK. Additionally, the proportion of revertant viruses inc reased progressively during the course of infection in these animals, and two of these animals developed fatal SAIDS. Taken together, these results demonstrated that in vivo selection for the ability of SIV Nef to associate with NAK was correlated with the induction of SAIDS. Acc ordingly, these studies implicate a role for the conserved SH3 ligand domain for Nef function in virally induced immunodeficiency.