IDENTIFICATION OF A MUTATION AT CODON-65 IN THE IKKK MOTIF OF REVERSE-TRANSCRIPTASE THAT ENCODES HUMAN-IMMUNODEFICIENCY-VIRUS RESISTANCE TO2',3'-DIDEOXYCYTIDINE AND 2',3'-DIDEOXY-3'-THIACYTIDINE

The technique of in vitro selection was used to generate variants of t he human immunodeficiency virus type 1 that are resistant to 2',3'-did eoxycytidine (ddC). Most of the pol regions of such viruses, including the complete reverse transcriptase open reading frame and portions of flanking protease and integrase genes, were cloned and sequenced, usi ng PCR-based procedures. Mutations were variously detected at amino ac id site 65 (Lys-->Arg; AAA-->AGA) and at a previously reported codon, site 184 (Met-->Val; ATG-->GTG). We introduced the site 65 mutation in to the pol gene of infectious, cloned HxB2-D DNA by site-directed muta genesis in order to confirm by viral replication assay the importance of this site in conferring resistance to ddC. The recombinant virus po ssessed greater than 10-fold resistance against this compound in compa rison with parental HxB2-D. Cross-resistance of approximately 20- and 3-fold, respectively, was detectable against the (-) enantiomer of 2', 3'-dideoxy-3'-thiacytidine and 2',3'-dideoxyinosine but not against 3' -azido-3'-deoxythymidine. Combinations of the site 65 and 184 mutation s did not yield levels of resistance higher than those attained with t he site 65 mutation alone. The presence of the site 65 mutation was co nfirmed by PCR analysis of peripheral blood mononuclear cells from pat ients on long-term ddC therapy in 4 of 11 cases tested. Viruses that p ossessed a ddC resistance phenotype were isolated from subjects whose viruses contained the site 65 mutation in each of four instances. Four of these clinical samples were also demonstrated to possess the Met-1 84-->Val mutation, and one of them possessed both the Lys-65-->Arg and Met-184-->Val substitutions. Direct cloning and sequencing revealed t he site 65 mutation in viruses isolated from these individuals.