Background:  Thymidine analogue-associated mutations (TAM) in HIV-1 reverse transcriptase (RT) can compromise clinical response to all NRTI. However, with the exception of zidovudine (AZT), increases in EC₅₀ values associated with these mutations are usually small in phenotypic assays. TAM were shown to enhance the excision of incorporated NRTI, which can only occur before the RT enzyme translocates to the next template position. Here we studied the availability of the pre-translocational state in dependence of both the sequence context and the chemical nature of the nucleotide analogue.

Methods:  Various primer/template combinations were scanned with translocation specific RT inhibitors for sites that exist predominantly pre-translocation. The translocation status was confirmed through high-resolution footprinting. Excision reactions were then carried out with defined sequences that show a bias toward pre- and post-translocation, respectively.

Results:  Primer/template combinations that favor the pre-translocational conformation exhibited higher rates of excision of 2’,3’-dideoxythymidine (ddTTP) when compared to substrates that show a bias toward post-translocation. The same trend is seen with both wild type RT and TAM containing mutant enzymes. In contrast, differences in rates of excision associated with pre- and post-translocated complexes are insignificant when the primer was terminated with AZT-monophosphate (MP). In this case, most of the sequences showed high levels of excision, regardless of a sequence-dependent bias of the translocational equilibrium. In multi-site excision assays, AZT-terminated sites behave collectively similarly, i.e. all are easily excised. In contrast, other inhibitors that are incorporated at the same positions show differential patterns of excision based on the variations in translocational equilibrium.

Conclusions:  The results of this study point to genome-wide differences in the translocation status of HIV-1 RT. The excision of AZT is less sensitive to these differences, when the efficiency of the reaction is compared with compounds that do not contain a 3’-azido group. Thus, AZT appears to abolish these differences, effectively making more sites more susceptible to excision, which helps to explain its relatively high phenotypic resistance.