SummaryTo investigate the function of the Vif protein of the simian immunodeficiency virus (SIV), mutations were introduced into the SIVmac239vifgene without affecting the reading frames of other overlapping genes. The phenotypes of these mutant viruses were examined with respect to viral replication and the expression and processing of viral proteins. Transfection of v/f-mutant proviral DNA into established T cell lines resulted in a significant delay in the onset of virus replication compared to that seen with the wild-type pro virus. The efficiency of replication of the v;/-mutant virus was dependent on cell type. MT-4 cells were permissive for replication of thevifmutant, while replication in CEMxl74 cells was severely restricted. Little or no virus replication was observed following cell-free infection of the CEMxl74 cell line and macaque peripheral blood mononuclear cells (PBMC). These results indicate that the requirement forvifduring the replication of SIVmac239 is dependent on cell type, as has been observed for HIV-1. Following cell-free infection, mutant viruses containing combined deletions invif andthe other regulatory genes (vpx, vpr, andnef) displayed replication kinetics similar to that of viruses containing the deletion ofvifalone. Viral protein expression and processing in MT-4 cells of vi/-deleted viruses were indistinguishable from those of the wild-type virus. The effects of two different point mutations invif wereexamined. One point mutant invifreverted to the genetic sequence of the wild-type virus within 2 weeks. A second point mutant that did not show genetic reversion displayed a similar kinetics of virus replication to that of thevifdeletion mutant. These data show that impaired replication ofvifmutant viruses results from mutation of thevifgene rather than the deletion of c/.?-acting elements, and also indicate that a strong selection exists against vi/-defective viruses.