Macaques infected with cloned simian immunodeficiency virus show recurring nef gene alterations

We compared nef gene sequences isolated by polymerase chain reaction from peripheral blood lymphocyte DNA of macaques that had been inoculated with either biologically (E11S) or molecularly (clone 8) cloned SIV/Mne. Two samples from each animal obtained either early (weeks 2-8) or late (weeks 21-137) after infection were analyzed. Three substitutions in the predicted Nef amino acid sequence were seen in all animals at the late time point, and two other substitutions were seen in all except one. Two of the common exchanges are located approximately 40 residues apart in the Nef core sequence but are juxtaposed on the tertiary structure as judged by computer modeling using the structure of the HIV Nef core protein as a guide. Most recurrent in vivo changes replaced a residue found in the cloned Nef sequence with one present in a consensus derived by aligning the Nef sequences of the SIV/Sm clade. Recombinant virus containing a macaque-adapted (MA nef) nef on the clone 8 backbone was 3-fold more infectious on SMAGI cells than the original virus. A lymphocyte line infected with SIV-clone 8-MAnef contained a large proportion of cells carrying provirus with defective nef genes. These findings suggest that the nef gene of the cloned SIV/Mne had undergone attenuating mutations during propagation in tissue culture that were "corrected" in vivo.