Background:  Mutations in the connection (A371V) and RNase H (Q509L) domains of HIV-1 reverse transcriptase (RT) are selected with thymidine analog mutations (TAM) D67N, K70R, and T215I/F by 3’-azido-3’-dideoxythymidine (AZT) in vitro and confer 50-fold increase in AZT resistance compared to TAM alone. Together, A371V/Q509L in combination with TAM decrease RNase H activity and increase AZT-monophosphate (AZT-MP) excision during multiple rounds of polymerization on an RNA template. We have now differentiated the individual roles of A371V and Q509L and clarified the link between RNase H activity and excision.
Methods:  AZT-MP excision during multiple rounds of incorporation was determined using a 214 nucleotide RNA or DNA template annealed to a radio-labeled DNA primer. RNase H cleavage product formation was assessed from an RNA/DNA template/primer. RNA/DNA duplexes were constructed to represent cleavage products and were used to evaluate single turnover AZT-MP excision.
Results:  Compared to TAM alone (D67N/K70R/T215F), TAM/A371V, TAM/Q509L, and TAM/A371V/Q509L increased AZT-MP excision 1.7-, 2.7-, and 2.9-fold, respectively, during multiple rounds of incorporation on a RNA template. There was little difference in the rate of deoxyribonucleotide triphosphate (dNTP) incorporation between enzymes and no increase in AZT-MP excision on a DNA template. In addition, TAM/A371V, TAM/Q509L, and TAM/A371V/Q509L decreased RNase H cleavage product formation 1.3-, 2.1- and 2.1-fold compared to TAM alone. The rate of AZT-MP excision under single turnover conditions on a 10 nucleotide RNA/DNA duplex increased 1.1-, 1.4-, and 1.3-fold, respectively, for TAM/A371V, TAM/Q509L, and TAM/A371V/Q509L, compared to TAM alone. No change in AZT-MP excision was observed on RNA/DNA duplexes of 12 nucleotides or longer.
Conclusions:  Q509L and A371V/Q509L with TAM impair formation of RNase H cleavage products, which increases AZT-MP excision on RNA/DNA duplexes by reducing template degradation. In addition, Q509L and A371V/Q509L increase the efficiency of excision on short RNA/DNA duplexes. A371V with TAM had little effect on AZT-MP excision or RNase H cleavage, thus its role remains undefined.