Second-site revertants of a simian immunodeficiency virus gp41 mutant defective in envelope glycoprotein incorporation

We previously characterized a series of small in-frame deletions within the C-terminal third of the simian immunodeficiency virus (SIV) gp41 cytoplasmic domain that significantly impair the incorporation of the envelope (Env) glycoprotein into particles and Env-mediated virus entry. Among these mutations, removal of Env residues 832-837 caused the most drastic defective phenotype. In the present study, we introduced the Δ832-837 deletion into the PBj1.9 molecular clone and investigated the effect of this env mutation on virus replication in the CEMx174 cell line. This in-frame deletion was found to severely compromise virus replication. Interestingly, long-term culture of the PBjEnvΔ832-837 mutant led to the emergence of two independent populations of revertant viruses that, while differing in the time point at which they appear, encode truncated gp41 cytoplasmic tails of similar lengths. The first emergent virus population contained a premature stop codon mutation at Env residue 778, whereas the late-appearing population harbored a stop codon mutation at Env residue 774, which results in the truncation of the gp41 cytoplasmic tail to 52 and 48 amino acids, respectively. Analysis of derivatives of PBjEnvΔ832-837 containing either the Tyr778stop or the Trp774stop mutations demonstrated that these second-site changes were sufficient to reverse the Env incorporation and infectivity defects imposed by the original Δ832-837 deletion, as well as to confer to the Env double mutants essentially wild-type replication kinetics. Our results thus provide further insight into the mechanisms underlying SIV adaptation to novel selective forces.