Kinetic studies of HIV-1 Reverse Transcriptase nucleotide selectivity, drug resistance and RNase H activity

Mechanisms of nucleotide selectivity and drug resistance by HIV-1 Reverse Transcriptase (HIVRT) were examined using rapid kinetic methods and global data fitting. Thymidine analog resistance mutations provide only two-fold discrimination against the incorporation of AZT-triphosphate; therefore, it is generally believed that resistance arises from nucleotide excision, where ATP reacts with the 3’ terminal base to produce a dinucleotide tetraphosphate and a 3’-OH terminated primer capable of subsequent extension. Single turnover kinetic analysis with global data fitting revealed the intrinsic rate and equilibrium constants governing each step leading to resistance to chain terminators. Our data suggested the net resistance to AZT arises from nearly equal contributions involving discrimination during incorporation (2x), enhanced ATP-dependent excision (2-5x), reduced binding of the next correct nucleotide (2.6-5x) and more favored binding of the DNA primer in the N-site (5-20x). Chemistry is generally the only rate-limiting step for product formation during DNA polymerization with a DNA template, but with an RNA template we show that pyrophosphate (PPi) release was rate-limiting. Due to the slow PPi release step, the rate of reversal of chemistry could also be determined affording the first measurement of the equilibrium constants governing polymerization and the first complete free energy profile for HIVRT. Although PPi release is rate-limiting, nucleotide binding remains as the specificity-determining step. However, PPi release becomes exceedingly slow following mis-incorporation and thereby contributes to the specificity constant. Our data demonstrate that the fidelity of HIVRT has been underestimated by >20-fold in the past 20 years since the slow PPi release has been overlooked. The rate-limiting PPi release allows synchronization and coordination of the polymerase and RNase-H activities. Studies were undertaken to examine proposed coordination between the polymerase and RNase-H activities. Direct, simultaneous measurement of the two activities established a mechanism by which polymerization and RNase-H activities are coordinated by working independently at comparable rates, but with different efficiencies. In contrast to polymerization, RNase-H requires ~4–6 base pairs extending beyond the active site for optimal reactivity, providing fast but infrequent cleavage events. Consequently, the polymerase and RNase-H activities are seamlessly coordinated without any direct communication between the two sites.