12th International HIV Drug Resistance Workshop 10–14 June 2003, Cabo del Sol, Los Cabos, Mexico

BACKGROUND: The phenotypic mechanism of HIV-1 resistance to 3´-azido-2´,3´-dideoxythymidie (AZT) has been proposed to involve the removal or excision of the incorporated chain-terminating AZT molecule by a reverse transcriptase (RT)-mediated enzymatic reaction termed phosphorolysis. Previous studies have indicated that primers terminated with AZTmonophosphate (AZT-MP) are better substrates for this reaction than those terminated with 2´-3´- dideoxynucleoside monophosphate (2´,3´-ddNMP) analogs that lack a 3´-azido moiety. This has lead to the suggestion that the 3´-azido group may be a major structural determinant for maintaining the primer terminus in the appropriate site to allow phosphorolytic excision by AZT-resistant RT. We tested this possibility by carrying out detailed biochemical and virological evaluations of the incorporation, phosphorolytic excision and antiviral properties of a panel of 3´-azido- 2´,3´-ddN compounds including 3´-azido-2´,3´-ddA, 3´- azido-2´,3´-ddC, 3´-azido-2´,3´-ddG, 3´-azido-2´,3´-ddU and AZT.

METHODS: Steady-state and pre-steady-state kinetic parameters for the incorporation and ATP-mediated excision of the 3´-azido-2´,3´-ddNTPs were determined for purified wild-type (wt) RT and D67N/K70R/ T215F/K219Q AZT-resistant RT. Antiviral activities of the 3´-azido-2´,3´-ddN nucleosides were evaluated in MT2 cells using WT or D67N/K70R/T215Y/K219Q AZT-resistant virus.

RESULTS: Each of the 3´-azido-2´,3´-ddNTPs was an excellent substrate for DNA-dependent single nucleotide incorporation reactions catalysed by wt and AZT-resistant RT. The relative catalytic efficiencies of incorporation (k_pol/K_d) were 3´-azido-2´,3´-ddA > AZT > 3´-azido-2´,3´-ddC, with no differences noted between wt and AZT-resistant RT. In contrast to the incorporation data, significant differences in the rates of ATP-mediated phosphorolytic excision of the various 3´-azido-2´,3´-ddNMP were noted. The relative rates of ATP-mediated phosphorolysis by wt and AZTresistant RT (in the absence of dNTP) were 3´-azido- 2´,3´-ddA>AZT>3´-azido-2´,3´-ddC>3´-azido-2´,3´- ddG. However, under reaction conditions that enabled multiple rounds of 3´-azido-2´,3´-ddNMP incorporation and excision, the AZT-resistant RT enzyme was 15-fold more efficient at forming full length DNA products in the presence of AZTTP than the wt enzyme. In contrast, only a threefold difference between mutant and wt RT was noted for reactions in the presence of 3-azido-2´,3´-ddCTP, and no differences between the two enzymes were noted for reactions in the presence of 3´-azido-2´,3´-ddATP and 3´- azido-2´,3´-ddGTP. The antiviral activities of the various 3´-azido-2´,3´-ddN were consistent with the enzymatic data, in that AZT-resistant virus was 10-fold resistant to AZT and fourfold resistant to 3´-azido- 2´,3´-ddC, but was not cross-resistant to either 3´- azido-2´,3´-ddA or 3´-azido-2´,3´-ddG.

CONCLUSIONS: AZT resistance mutations do not confer significant cross resistance of RT or virus to other nucleosides having a 3´-azido group. Furthermore, the presence of a 3´-azido group on the 3´-terminal nucleotide of the primer does not enhance phosphorolytic excision by AZT-resistant RT in vitro, suggesting that other structural factors must play a role in defining the specificity of the excision phenotype arising from mutations correlated with AZT-resistance.

PRESENTING AUTHOR: N Sluis-Cremer

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2003-07-08
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