AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 20, Number 7, 2004, pp. 733–741 © Mary Ann Liebert, Inc. Second-Site Revertants of a Simian Immunodeficiency Virus gp41 Mutant Defective in Envelope Glycoprotein Incorporation CRISTINA C. P. CELMA, 1 JULIETA M. MANRIQUE, 1 ERIC HUNTER, 2 JOSÉ L. AFFRANCHINO, 1 and SILVIA A. GONZÁLEZ 1 ABSTRACT We previously characterized a series of small in-frame deletions within the C-terminal third of the simian im- munodeficiency virus (SIV) gp41 cytoplasmic domain that significantly impair the incorporation of the enve- lope (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 832–837 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 replica- tion. Interestingly, long-term culture of the PBjEnv832–837 mutant led to the emergence of two indepen- dent populations of revertant viruses that, while differing in the time point at which they appear, encode trun- cated 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 mu- tation 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 PBjEnv832–837 containing either the Tyr778stop or the Trp774stop mutations demonstrated that these second-site changes were sufficient to reverse the Env incor- poration and infectivity defects imposed by the original 832–837 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. 733 INTRODUCTION T HE ENTRY OF THE HUMAN AND SIMIAN IMMUNODEFICIENCY VIRUSES (HIV and SIV, respectively) into their target cells constitutes the initial step in the replication cycle of these viruses. This process is mediated by molecular interactions be- tween the viral envelope (Env) glycoprotein and the two com- ponents of the cell receptor complex, CD4 and a chemokine receptor. Both HIV and SIV Env glycoproteins are first syn- thesized as a single heavily glycosylated precursor that is pro- teolytically cleaved by a cellular protease during transport to the cell surface. 1 Cleavage of the Env precursor results in a functional complex composed of the surface (SU) glycoprotein gp120 noncovalently associated with the transmembrane (TM) glycoprotein gp41. The SU glycoprotein binds to the CD4 and chemokine receptors, whereas the TM subunit mediates fusion of the viral and cellular membranes. 2 The TM glycoproteins of SIV and HIV contain an N-terminal extracellular domain, a membrane-spanning domain, and a C-terminal intracellular do- main with an endocytosis/basolateral targeting signal and two amphipathic regions. 1 The cytoplasmic domain of the Env pro- teins of these primate lentiviruses is exceptionally long com- pared with those of other retroviruses: 164 and about 150 amino acids for SIV and HIV-1, respectively. The biological function of this long cytoplasmic domain has been the subject of sev- eral studies. In this regard, it has been shown that the SIV TM cytoplasmic domain is implicated in viral functions, such as regulation of Env expression at the cell surface 3,4 and modula- tion of fusogenicity and infectivity. 5–10 We have recently analyzed the role of the SIV TM cyto- 1 Centro de Virología Animal (CEVAN-CONICET), Serrano 669, (C1414DEM) Buenos Aires, Argentina. 2 Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294.