Abstract
In vitro selection in MT-4 cells was used to generate human immunodeficiency virus-type 1 (HIV 1) variants that are resistant to 2',3'-dideoxycytidine (ddC), 2',3'-didcoxyinosine (ddI) and the (-) enantiomer of 2' ,3'-dideoxy-3'-thiacytidine (3TC). The complete reverse transcriptase open reading frames of these viruses, and portions of flanking protease and integrase within the pol gene, were cloned and sequenced by polymerase chain reaction (PCR) techniques. Mulalions were observed at each of amino acid sites 65 (Lys → Arg: AAA → AGA) and 184 (Met → Val: ATG → GTG) when ddC was used in this protocol, and at site 184 only when either 3TC or ddl was employed. These mutations were introduced into the pol gene of infectious recombinant HXB2-D DNA by site-directed mutagenesis to confirm, by viral replication assay, their importance in conferring resistance against these drugs. A recombinant virus containing the site 65 mutation only possessed greater than 10-fold resistance against ddC compared with parental HXB2-D. Moreover, cross-resistance of about 20-fold and threefold, respectively, was delectable against 3TC and dell but not against 3'-azido-3'-deoxthymidine (AZT). When the 65 and 184 mutations were combined into HXB2-D, the resultant construct did not possess higher levels of resistance lo any of these drugs than observed with the site 65 or 184 mutation alone. These mutations were further demonstrated by PCH analysis of peripheral blood mononuclear cells from 10 patients on long term ddC therapy, although variable patterns were observed in terms of which of the two mutations or both were present. Sometimes, the wild-type site 65 codon was also detected, indicating the presence of mixtures of viral quasi-species. Direct cloning and sequencing revealed the site 65 mutation in viruses isolated from patients on prolonged ddC therapy.