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J. Biol. Chem., Vol. 281, Issue 38, 27744-27752, September 22, 2006

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Selective Excision of Chain-terminating Nucleotides by HIV-1 Reverse Transcriptase with Phosphonoformate as Substrate^*

From the Departamento de Bioquímica y Biología Molecular, Universidad de Navarra, Calle Irunlarrea s/n, 31008 Pamplona, Spain

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP_i) analogs on PP_i- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP_i- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT·template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP_i or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

Received for publication, April 7, 2006 , and in revised form, July 5, 2006.

^* This work was supported, in part, by a grant from the Departamento de Educación y Cultura from the Gobierno de Navarra and by a joint grant from the Gobierno de Navarra and the Conseil Regional d'Aquitaine (Fondo Común de Cooperación Navarra-Aquitania). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Departamento de Bioquímica y Biología Molecular, Universidad de Navarra, Calle Irunlarrea s/n, 31008 Pamplona, Spain. Tel.: 34-948425600; Fax: 34-948425649; E-mail: jjmirujo{at}unav.es.

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