Background:  Thymidine analogue associated mutations (TAM) in reverse transcriptase (RT) cause resistance to zidovudine (AZT) and other nucleoside analogue RT inhibitors by increasing the rate of excision. Recent findings have identified mutations in the connection domain that augment AZT resistance, usually in a background of TAM. It has been suggested that these connection mutations may compromise RNase H cleavage, providing more time for AZT excision to occur. However, the mechanism underlying the selective increase in AZT resistance remains elusive. 

Methods:  In this study, we used connection mutations A360V and N348I as a model to address this problem with a combination of complementary biochemical tools. Previous studies revealed that TAM in combination with A360V and N348I are associated with marked decreases in phenotypic susceptibility to AZT, while susceptibility to stavudine (d4T) remains relatively low. Here we characterized and compared N348I, A360V, TAM, and TAM/N348I/A360V mutant enzymes to delineate the underlying mechanism.

Results:  Initial experiments confirm that N348I and A360V in combination with TAM, decrease the efficiency of RNase H cleavage and increase the amount of rescued reaction product following ATP-dependent excision. Band-shift assays show that the TAM/N348I/A360V mutant can form a stable binary complex with AZT-terminated RNA/DNA substrates, while RNase H cleavage with wild type RT yield 7- to 8-mer single-stranded DNA primers that do not bind to the enzyme. The TAM/N348I/A360V mutant accumulates stable 10- to 12-mer hybrids that can rebind to RT. The short hybrids dissociate from the RNase H-competent complex and later rebind to RT in a polymerase-competent mode that allows excision to occur. Complexes containing the short, transiently formed hybrids are distinguishable from complexes with the intact substrate. The same effect is seen with d4T-terminated primers; however, the formation of a dead-end complex with the incoming nucleotide blocks excision in this case, which diminishes the benefits of connections mutations. In accordance with band-shift experiments, measurements of rates of dissociation reveal that N348I and A360V promote dissociation selectively from an RNase H-competent complex.

Conclusions:  Selective dissociation of transiently formed hybrids from RNase H-competent complexes provides a mechanism for the selective increase in AZT resistance associated with connection mutations N348I and A360V.