Document 0619
 DOCN  M9550619
 TI    Sequence analysis of proviral HIV RT amplified directly by a
       semi-quantitative technique from AZT treated patients.
 DT    9505
 AU    Stein CA; Levantis P; Oxford JS; Department of Medical Microbiology,
       London Hospital Medical; College, England.
 SO    J Med Virol. 1994 Oct;44(2):115-21. Unique Identifier : AIDSLINE
       MED/95156015
 AB    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 National
       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 (Ile), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196
       (Glu), 200 (Ile), 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.
 DE    Amino Acid Sequence  Base Sequence  Drug Resistance, Microbial/GENETICS
       DNA Primers/GENETICS  DNA, Viral/GENETICS  Human  HIV Infections/DRUG
       THERAPY/*VIROLOGY  HIV-1/DRUG EFFECTS/*ENZYMOLOGY/*GENETICS  Leukocytes,
       Mononuclear/VIROLOGY  Molecular Sequence Data  Point Mutation
       Polymerase Chain Reaction  Proviruses/ENZYMOLOGY/GENETICS  Reverse
       Transcriptase/*GENETICS  Time Factors  Zidovudine/THERAPEUTIC USE
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).