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 substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophage-tropic, 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. Acitve-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 pot gene can tolerate many silent mutations in the polymerase domain without affecting the functionality of the HIV-1 genome. The system was validated biochemically and virologically using the V75T substitution associated with stavudine resistance. (C) 2001 Academic Press.