THE ROLE OF UPSTREAM U3 SEQUENCES IN THE PATHOGENESIS OF SIMIAN IMMUNODEFICIENCY VIRUS-INDUCED AIDS IN RHESUS-MONKEYS

The nef reading frame overlaps about 70% of the U3 region of the 3' lo ng terminal repeat (LTR) in primate lentiviruses. We investigated the functional role of these overlapping U3 sequences by analyzing the pro perties of three mutant forms of the pathogenic SIVmac239 clone. In mu tant UScon, 90 of 275 bp in the upstream sequences (US) of U3 were cha nged in a conservative fashion without changing the predicted nef codi ng sequence. In mutant USnon, 101 of 275 bp in this region were change d ina nonconservative fashion, again without changing the predicted ne f coding sequence. In mutant Delta US, 275 bp in this region were dele ted. Full-size, immunoreactive nef protein was synthesized in cells in fected with the UScon and USnon mutants. The USnon and AUS mutants rep licated with similar kinetics and to similar extents as wild-type, par ental STVmac239 in primary rhesus monkey peripheral blood mononuclear cell (PBMC) cultures. The UScon mutant replicated with slightly delaye d kinetics in rhesus monkey PBMC cultures. In the CEMx174 cell line, t he Delta US mutant replicated similarly to the wild type, but the USco n and USnon mutants replicated with significantly delayed kinetics. An alysis of LTR-driven chloramphenicol acetyltransferase (CAT) activity and the effects of 5-azacytidine on virus replication suggested that t he growth defect of the point mutants in CEMx174 cells was due in whol e or in part to the introduction of multiple CG methylation sites in p roviral DNA. Rhesus monkeys were experimentally infected with the USco n and USnon mutants, and the characteristics of the infection were com pared,vith those of the parental STVmac239. Analysis of the levels of plasma antigenemia, virus load, and CD4(+) cells in PBMC revealed no d ecreased virulence of the mutant viruses. Analysis of lymph node biops ies taken from animals that received mutant viruses revealed histologi c changes and levels of virus expression indistinguishable from those of the wild type. Furthermore, the wild-type behavior of the mutant vi ruses in rhesus monkeys occurred without any specific reversional even ts through at least 20 weeks of infection. These results, and the rece nt results of Kirchhoff et al. (F. Kirchhoff, H. W. Kestler III, and R . C. Desrosiers, J. Virol. 68:2031-2037, 1994), suggest that these ups tream sequences in U3 are primarily or exclusively nef coding sequence .