An integrated system to study multiply substituted human immunodeficiency virus type 1 reverse transcriptase

Analytical Biochemistry
Joëlle BorettoBruno Canard

Abstract

We describe a gene system allowing the facile production of multiply substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophagetropic, non-laboratory-adapted human immunodeficiency virus type 1 (HIV-1) AD8 strain. Thirteen unique silent restriction sites were introduced in the pol gene encoding HIV-1 RT, allowing easy introduction of mutations. To simplify genetic manipulation and generate p66/p51 heterodimers in Escherichia coli, a gene construct of the viral protease alone was optimized for expression from a separate vector carrying a p15A origin of replication. Active-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzyme than that obtained by alternate methods. To facilitate phenotype/genotype correlations, the modified RT gene was designed to be easily reintroduced into a recombinant proviral AD8 HIV-1 DNA. Infectious viruses made from this vector were undistinguishable from wild-type AD8 HIV-1, an isolate able to infect peripheral blood mononuclear cells and macrophages. Thus, the pol gene can tolerate many silent mutations in the p...Continue Reading

References

Jan 26, 1990·European Journal of Biochemistry·S F Le Grice, F Grüninger-Leitch
Apr 16, 1990·Biochemical and Biophysical Research Communications·M C GravesF Grüninger-Leitch
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Mar 28, 2012·Antimicrobial Agents and Chemotherapy·A M L Oude LashofB J Kullberg

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Citations

Aug 17, 2011·The Journal of Infectious Diseases·Roger ParedesUNKNOWN EuroSIDA Study Group
Feb 22, 2012·The FEBS Journal·Verónica BarrioluengoLuis Menéndez-Arias
Aug 5, 2020·Antimicrobial Agents and Chemotherapy·Moisés A ÁrquezLuis Menéndez-Arias

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