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M9490505.TXT
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1994-09-24
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Document 0505
DOCN M9490505
TI Preferential incorporation of nucleoside analogs after template
switching during human immunodeficiency virus reverse transcription.
DT 9411
AU Arts EJ; Wainberg MA; McGill AIDS Centre, Lady Davis Institute-Jewish
General Hospital,; Montreal, Quebec, Canada.
SO Antimicrob Agents Chemother. 1994 May;38(5):1008-16. Unique Identifier :
AIDSLINE MED/94346798
AB We assessed the effects of 3'-azido-3'-deoxythymidine (AZT),
2',3'-dideoxyinosine (ddI), and the (-) enantiomer of
2',3'-dideoxy-3'-thiacytidine (3TC) on reverse transcription in
CD4-positive cells by isolating truncated human immunodeficiency virus
(HIV) DNA fragments. Jurkat cells were treated with AZT (2 microM), ddI
(200 microM), or 3TC (50 microM) prior to infection with HIV.
Low-molecular-weight DNA was isolated and amplified by PCR with primer
pairs which identify different segments of HIV proviral DNA. We found
that the HIV DNA fragments generated from drug-treated, HIV-exposed
Jurkat cells were truncated at a ratio of 15:1 [i.e., (-) strong-stop
DNA to HIV DNA generated after the first template switch]. Full-length
DNA was observed in the case of untreated, HIV-infected cultures.
Following nucleoside analog treatment of HIV-exposed Jurkat cells,
reverse transcription was terminated only after the synthesis of (-)
strong-stop DNA. The nucleoside analogs tested, i.e., AZT, ddI, and 3TC,
preferentially chain terminated viral DNA synthesis immediately
following the first template switch. The (-) strong-stop HIV DNA was
present in AZT-treated and untreated cultures for at least 6 days. We
also carried out cell-free reverse transcription/template-switching
reactions involving tRNA(Lys3) or a deoxyoligonucleotide as a primer, as
a means of studying the selective incorporation of AZT triphosphate into
proviral DNA. When reactions were primed with tRNA(Lys3), we found that
AZT triphosphate was preferentially incorporated after template
switching.
DE Antiviral Agents/METABOLISM Base Sequence Cell Line
Didanosine/METABOLISM DNA, Viral/ANALYSIS/ISOLATION & PURIF Human
HIV-1/GENETICS/*METABOLISM Molecular Sequence Data
Nucleosides/*METABOLISM Polymerase Chain Reaction RNA, Transfer,
Lys/METABOLISM Support, Non-U.S. Gov't Templates Thymine
Nucleotides/METABOLISM *Transcription, Genetic T4 Lymphocytes/DRUG
EFFECTS/METABOLISM/MICROBIOLOGY Zidovudine/ANALOGS &
DERIVATIVES/METABOLISM JOURNAL ARTICLE
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).