3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase

Abstract

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rC(m))-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. K(m) and K(i) values of 2.8 and 0.04 μM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 μM, respectively, with activated calf thymus DNA and 0.3 and 0.01 μM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases α and β was considerably weaker. The K(m) and K(i) values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 μM, respectively, for DNA polymerase α and 6.0 and 73 μM, respectively, for DNA polymerase β. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.