13th International HIV Drug Resistance Workshop


8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain


SELECTIVE PRIMER UNBLOCKING OF CARBOVIR, LAMIVUDINE, TENOFOVIR AND ZIDOVUDINE BY WILD-TYPE HIV REVERSE TRANSCRIPTASE WITH NUCLEOTIDE AND DEOXYNUCLEOTIDE TRIPHOSPHATES

Antiviral Therapy 2004; 9:S30

P Gerondelis, MR Underwood and ER Lanier
Department of International Clinical Virology, GlaxoSmithKline, Research Triangle Park, NC, USA

BACKGROUND: Incorporation of nucleoside and nucleotide reverse transcriptase inhibitors (NRTI/ NtRTIs) by HIV reverse transcriptase (RT) into primer-template results in chain-termination of DNA synthesis. One type of resistance to the NRTI class is the nucleophilic-excision primer unblocking (PUB) activity of RT, which may compromise the stability of chain termination. Studies of the PUB activity of RT have primarily focused on the purine nucleoside triphosphate (NTP), ATP, as the nucleophilic PUBsubstrate. With the exception of ddAMP-terminated primer-template, NRTI/NtRTIs have not been evaluated for their sensitivity to the pyrimidine-NTPs, CTP and UTP. In addition, deoxynucleoside triphosphates (dNTPs) have not been generally studied as substrates for PUB.

OBJECTIVE: To evaluate the PUB of NRTI/NtRTIs by measuring sensitivity to the NTP- and dNTP-mediated PUB activity of wild-type (wt) RT.

METHODS: Wt RT PUB activity was evaluated with primer-templates chain-terminated by carbovir-MP, lamivudine-MP, tenofovir and zidovudine-MP. NTPs (ATP, CTP, GTP and UTP), and dNTPs (dATP, dCTP, dGTP and dTTP) were used as nucleophiles in the PUB reaction.

RESULTS: Carbovir-MP and zidovudine-MP terminated primer-templates were susceptible, while lamivudine was resistant to PUB with NTPs as the nucleophiles. Interestingly, the tenofovir-terminated primer-template was susceptible only to pyrimidine (i.e. CTP and UTP) NTP-based PUB. In addition, tenofovir-terminated primer-template was susceptible to dNTPPUB, similar to that observed in studies of ddAMPterminated primer-template (P Meyer et al.; Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13471-6). In contrast, the C-, G- and T-based NRTIs of this study were relatively resistant to dNTP-PUB.

CONCLUSIONS: These studies suggest that NRTIs/ NtRTIs have different capacities to maintain chaintermination of wt RT catalysed DNA-dependent DNA polymerization under different experimental conditions, dependent in part upon the structure of the pyrophosphate donor. Generally, the PUB activity of wt RT appears to be dependent upon the nucleophile, the NRTI/NtRTI and the primer-template. Future studies of the PUB activity of both wt and resistant RT should take into consideration the selective PUB by nucleophiles other than ATP, as well as the dependence of this activity on specific primer-template sequence.

PRESENTING AUTHOR: ER Lanier

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2004-06-08
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