SEQUENCE-ANALYSIS OF PROVIRAL HIV RT AMPLIFIED DIRECTLY BY A SEMIQUANTITATIVE TECHNIQUE FROM AZT TREATED PATIENTS

To minimise possible arbitrary selective effects of culturing HIV, pro viral RT DNA was isolated directly from PBMCs of four patients treated for 6-14 months with AZT. RT DNA was amplified by PCR and sequenced d irectly without further in vitro manipulation. Eighteen changes additi onal to those 4 or 5 changes previously shown by genetic reconstructio n experiments [Kellam et al.: Proceedings of the National Academy of S ciences of the United States of America 89:1934-1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in tw o defined areas of the RT, from amino acids 60 to 70 and from 180 to 2 20. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients h arbouring resistant strains of HIV-1 were found in positions 60 (Vat), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 ( lie), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (lie) , 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs w ould be present in a single clinical sample. Therefore end point dilut ion PCR methodology was used, which allowed sequence analysis of separ ate proviral DNA molecules from the patients' proviral DNA. Even in pa tients who had received AZT for more than 10 months wild-type ''AZT-se nsitive'' RTs co-existed with mutated ''AZT-resistant'' RTs in the Sam e patient sample. (C) 1994 Wiley-Liss, Inc.