Loss of the SIVsmmPBj14 Phenotype andnefGenotype during Long-Term Survival of Macaques Infected by Mucosal Routes

Abstract The ability of the simian immunodeficiency virus SIVsmmPBj14 (SIV-PBj14) to activate and induce proliferation of quiescent peripheral blood lymphocytes from macaques is an in vitro correlate of its acutely lethal in vivo phenotype. SIV-PBj14 differs from other SIV strains by encoding tyrosine at amino acid 17 (Y17) in Nef, which generates an activation motif important for signal transduction. Although intravenous inoculation of pig-tailed macaques with SIV-PBj14 uniformly leads to death within 2 weeks, inoculation by mucosal routes results in persistent infections that progress to AIDS. In the present study, we determined whether viruses in long-term survivors retained not only the Nef Y17 residue but also the biologic properties associated with rapid disease and death. Viruses reisolated at early and late times after mucosal infection of macaques with SIV-PBj14 were tested in vivo for acute lethality and in vitro for the ability to replicate in and induce activation and proliferation of quiescent macaque lymphocytes. In addition, the coding sequence for the first 55 amino acids in Nef was amplified from proviral DNA or plasma virion RNA by PCR or RT-PCR, respectively, and nucleotide sequences were obtained. The results showed that the majority of the quasispecies that persisted as disease progressed not only lost biological properties unique to SIV-PBj14, but also lost through mutation either Y17 or Y28 in Nef, which together were part of the activation motif. In the case of Y17, these mutations were stepwise to histidine then arginine, the amino acid encoded in this position in other SIV strains. We conclude, therefore, that replicative properties of the acutely lethal virus provide no selective advantage during long-term infections with SIV-PBj14 and that disruption of the activation motif in Nef is associated with loss of the acutely lethal phenotype.