Simian immunodeficiency virus containing mutations in N-terminal tyrosine residues and in the PxxP motif in Nef replicates efficiently in rhesus macaques

SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a pu tative SH3-ligand domain (P(104)xxP(107)). In the present study, we investi gated the effects of combined mutations in these tyrosine and proline resid ues on simian immunodeficiency virus (SIV) Nef interactions with the cellul ar signal transduction and endocytic machinery. We found that mutation of Y 28F, Y39F, P(104)A and P(107)A (FFAA-Nef) had little effect on Nef function s such as the association with the cellular tyrosine kinase Src, downregula tion of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef va riant showed high viral loads during the acute phase of infection. Reversio ns in the mutated prolines were observed between 12 and 20 weeks postinfect ion. Importantly, reversion of A(107)-->P, which restored the ability of Ne f to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did n ot precede the development of a high viral load. The Y-28/Y-39-->F-28/F-39 substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt majo r aspects of SIV Nef function in vivo.