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

BACKGROUND: Phosphonoformate (PFA, foscarnet) binds at or in close proximity to the nucleotide binding site of HIV-1 reverse transcriptase (RT) and inhibits DNA synthesis presumably through interference with the exchange of pyrophosphate. Like other PFA-resistance conferring mutations, E89K is distal from the dNTP binding site and interacts with the template strand. This mutation is also associated with increased susceptibility to zidovudine (ZDV). We hypothesized that this effect might be attributable to alterations with regard to the precise alignment of ATP, that acts as a pyrophosphate donor and the primer/template substrate.

METHODS: To address this problem, we developed novel site-specific footprinting techniques that allowed us to monitor the position of RT on its template at single nucleotide resolution. We used different sources of hydroxyl radicals that promote site-specific cleavage on the bound template, and compared the cleavage patterns between wild-type RT and the mutant enzyme under different reaction conditions.

RESULTS: Wild-type RT promotes cleavage at positions –8 and –18 in the absence of the incoming dNTP, when using a substrate that contained a ZDV-terminated primer. This is the configuration that allows excision of the incorporated ZDV-monophosphate. The presence of the next dNTP forces the enzyme to translocate a single position further downstream, as evidenced by cleavage at positions –7 and –17. The presence of PFA diminishes the translocation of RT, which provides a novel mechanism for drug action. The E89K mutation appears to alter the relative position of RT. The mutant promotes cleavage at positions –9 and –19 in the absence of the incoming dNTP. Excision of ZDV cannot occur in this configuration, which helps to explain earlier findings that pointed toward diminished rates of primer unblocking associated with resistance to PFA. Moreover, relatively high concentrations of the next nucleotide are required to force the translocation of RT.

CONCLUSIONS: Enzymes containing PFA resistance conferring mutations alter the precise positioning of RT on its nucleic acid substrate. Such displacement diminishes the unblocking of ZDV-terminated primer strands. The high concentrations of dNTPs that are required to force translocation also helps to explain the diminished rates of DNA synthesis. These parameters may directly correlate with ZDV resensitization effects and diminished viral replication fitness associated with viruses that contain the E89K mutation.

PRESENTING AUTHOR: M Götte

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2003-07-08
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