Question

In the AZT Resistance paper by Walter Scott’s team they were able to provide a model from their data that supported the fact that pyrophosphorlysis is the most likely mechanism by which AZT is removed from the primer. Discuss (i) the difference between the WT and the AZT resistant mutant in how they remove the AZT terminated primer; (ii) additionally, discuss why ATP would be the logical choice as to the substrate used for AZT removal over PPi.

Answer 1

i) Unlike most cellular DNA polymerases, the reverse transcriptase (RT) of HIV-1 readily incorporates nucleotide analogs lacking a 3′OH group into the growing DNA chain. Once incorporated, these nucleotides block further elongation of the primer leading to inhibition of viral replication. Inhibition could be mitigated by an effective mechanism to remove the chain-terminating residue.

According to Walter Scott's model, Mutations in HIV-1 reverse transcriptase (RT) give rise to 3′-azido-3′-deoxythymidine (AZT) resistance by a mechanism that has not been studied before. This model shows that mutant RT has increased ability to remove AZTMP from blocked primers through a nucleotide-dependent reaction, producing dinucleoside polyphosphate and extendible primer. In the presence of physiological concentrations of ATP, mutant RT extended 12% to 15% of primers past multiple AZTMP termination sites versus less than 0.5% for wild type(WT). This is said to be the main difference between WT and AZT resistant mutant in the removal of AZT terminated primer.

ii) There is general agreement that ATP-dependent excision is significantly elevated in these mutants. Comparison of ATP-dependent excision activity between various mutant RTs has provided a strong biochemical correlate for AZT resistance measured by infectivity assays over a 150-fold range of excision activities. ATP is likely to be the most biologically relevant acceptor substrate for the excision reaction based on molecular modeling studies and the fact that the concentration of ATP is greater than that of other potential acceptor substrates in most living cells and another disadvantage of ppi is PPi accumulates during metabolic stimulation of lymphoid cells and PPi-dependent excision occurs much more rapidly than ATP-dependent excision, suggesting that the PPi-dependent reaction may also play a role in AZT susceptibility under some circumstances.

Since PPi-dependent excision regenerates the triphosphate form of the chain-terminating nucleotide, it is possible that the excision product will be reincorporated before it can be released from the surface of the enzyme or it may be released and immediately rebinds leading to reincorporation, this is the main reason as to why ATP remains the most widely used substrate in removal of AZT over PPi.