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J. Biol. Chem., Vol. 279, Issue 23, 24569-24577, June 4, 2004

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Molecular Determinants of Multi-nucleoside Analogue Resistance in HIV-1 Reverse Transcriptases Containing a Dipeptide Insertion in the Fingers Subdomain

EFFECT OF MUTATIONS D67N AND T215Y ON REMOVAL OF THYMIDINE NUCLEOTIDE ANALOGUES FROM BLOCKED DNA PRIMERS^*

Tania Matamoros, Sandra Franco, Blanca M. Vázquez-Álvarez, Antonio Mas¶, Miguel Ángel Martínez, and Luis Menéndez-Arias||

From the Centro de Biologìa Molecular "Severo Ochoa," Consejo Superior de Investigaciones Cientìficas-Universidad Autónoma de Madrid, 28049 Madrid, Spain and the Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Badalona, 08916 Barcelona, Spain

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA·DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.

Received for publication, November 19, 2003 , and in revised form, March 17, 2004.

^* This work was supported in part by FIPSE Grant 36207/01, Fondo de Investigación Sanitaria Grants 01/0067-01, 01/0067-02, and G03/173 ("Red Temá;tica Cooperativa de Investigación en SIDA"), and an institutional grant from Fundación Ramón Areces. 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.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table.

^¶ Present address: Facultat de Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, 08002 Barcelona, Spain.

^|| To whom correspondence should be addressed. Tel.: 34-914978477; Fax: 34-914974799; E-mail: lmenendez{at}cbm.uam.es.

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