AN INTEGRATION-DEFECTIVE U5 DELETION MUTANT OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERTS BY ELIMINATING ADDITIONAL LONG TERMINAL REPEAT SEQUENCES

Nonoverlapping deletions that eliminated the 5' (HIV-1(U5/603del)), mi ddle (HIV-1(U5/206del)), and 3' (HIV-1(U5/604del)) thirds of the U5 re gion of the human immunodeficiency virus type I (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (tran sient transfection of HeLa cells) and infectivity (T-cell lines and pe ripheral blood mononuclear cells). All three mutants exhibited a wild- type phenotype in directing the production and release of virus partic les from transfected HeLa cells. In infectivity assays, HIV-1(U5/206de l) was usually indistinguishable from wild-type virus whereas HIV-1(U5 /603del) was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1(U5/603del) particles re vealed a packaging defect resulting in a 10-fold reduction of encapsid ated genomic RNA. The HIV-1(U5/604del) mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type a nd multiplicity of infection. Quantitative competitive PCR indicated t hat HIV-1(U5/604del) synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1(U5/604del) mutant that displayed rapid infectio n kinetics emerged. Nucleotide sequence analysis indicated that the or iginal 26-nucleotide deletion present in HIV-1(U5/604del) had been ext ended an additional 19 nucleotides in the revertant virus. Characteriz ation of the HIV-1(U5/604del) mutant LTR in in vitro integration react ions revealed defective 3' processing and strand transfer activities t hat were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutan t virus.