Abstract
Human immunodeficiency virus (HIV) reverse transcriptase substitutes for temperature-sensitive DNA polymerase I (Pol Its) in Escherichia coli, providing a screen for anti-HIV reverse transcriptase nucleoside analogs in bacteria. Since phosphorylation of nucleosides in E. coli is limited to thymidine and its derivatives, we coexpressed herpes simplex virus thymidine kinase, an enzyme that phosphorylates a wide variety of nucleoside analogs, together with HIV reverse transcriptase. Coexpression of herpes simplex virus thymidine kinase and HIV reverse transcriptase rendered Pol Its cells sensitive to dideoxycytidine. Studies with different nucleoside analogs indicate that this bacterial screening system is able to select and identify nucleoside analogs that specifically target HIV reverse transcriptase.
Publication types
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Comparative Study
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Antiviral Agents / pharmacology*
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Cloning, Molecular
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Cytarabine / pharmacology*
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DNA Polymerase I
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Drug Evaluation, Preclinical / methods
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Escherichia coli / drug effects*
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Escherichia coli / enzymology
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Gene Expression
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HIV / enzymology*
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Kinetics
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Phosphorylation
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RNA-Directed DNA Polymerase / biosynthesis
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RNA-Directed DNA Polymerase / metabolism
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Recombinant Proteins / antagonists & inhibitors
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / metabolism
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Reverse Transcriptase Inhibitors*
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Simplexvirus / enzymology*
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Thymidine Kinase / antagonists & inhibitors*
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Thymidine Kinase / biosynthesis
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Thymidine Kinase / metabolism
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Zalcitabine / pharmacology*
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Zidovudine / pharmacology*
Substances
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Antiviral Agents
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Recombinant Proteins
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Reverse Transcriptase Inhibitors
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Cytarabine
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Zidovudine
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Zalcitabine
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Thymidine Kinase
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RNA-Directed DNA Polymerase
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DNA Polymerase I