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

We describe a gene system allowing the facile production of multiply substi tuted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic back ground of the macrophage-tropic, non-laboratory-adapted human immunodeficie ncy virus type 1 (HIV-1) AD8 strain. Thirteen unique silent restriction sit es were introduced in the pol gene encoding HIV-1 RT, allowing easy introdu ction of mutations. To simplify genetic manipulation and generate p66/p51 h eterodimers in Escherichia coli, a gene construct of the viral protease alo ne was optimized for expression from a separate vector carrying a p15A orig in of replication. Acitve-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzym e 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 thi s vector were undistinguishable from wild-type AD8 HIV-1, an isolate able t o infect peripheral blood mononuclear cells and macrophages, Thus, the pot gene can tolerate many silent mutations in the polymerase domain without af fecting the functionality of the HIV-1 genome. The system was validated bio chemically and virologically using the V75T substitution associated with st avudine resistance. (C) 2001 Academic Press.