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Sequence analysis of proviral HIV RT amplified directly by a semi‐quantitative technique from AZT treated patients
Author(s) -
Stein Christian A.,
Levantis P.,
Oxford J. S.
Publication year - 1994
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.1890440203
Subject(s) - virology , zidovudine , dna , point mutation , amino acid , human immunodeficiency virus (hiv) , biology , virus , microbiology and biotechnology , in vitro , provirus , mutation , sequence (biology) , genetics , viral disease , gene , genome
To minimise possible arbitrary selective effects of culturing HIV, proviral 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 directly without further in vitro manipulation. Eighteen changes additional to those 4 or 5 changes previously shown by genetic reconstruction experiments [Kellam et al.: Proceedings of the Nationa Academy of Sciences of the United States of America 89:1934–1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in two defined areas of the RT, from amino acids 60 to 70 and from 180 to 220. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients harbouring resistant strains of HIV‐1 were found in positions 60 (Val), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 (lle), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (lle), 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs would be present in a single clinical sample. Therefore end point dilution PCR methodology was used, which allowed sequence analysis of separate proviral DNA molecules from the patients' proviral DNA. Even in patients who had received AZT for more than 10 months wild‐type “AZT‐sensitive” RTs co‐existed with mutated “AZT‐resistant” RTs in the same patient sample. © 1994 Wiley‐Liss, Inc.