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Journal of Virology, January 2005, p. 812-822, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.812-822.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Quantification of the Effects on Viral DNA Synthesis of Reverse Transcriptase Mutations Conferring Human Immunodeficiency Virus Type 1 Resistance to Nucleoside Analogues

Francine Bouchonnet,1 Elisabeth Dam,2 Fabrizio Mammano,1 Vaea de Soultrait,1 Gaëlle Henneré,3 Henri Benech,3 François Clavel,1 and Allan J. Hance1*

INSERM U.552, Hôpital Bichat-Claude Bernard,1 Viralliance, Paris,2 Service de Pharmacologie et d'Immunologie, CEA, Gif-sur-Yvette, France3

Received 29 May 2004/ Accepted 3 September 2004

Human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) resistance mutations reduce the susceptibility of the virus to nucleoside analogues but may also impair viral DNA synthesis. To further characterize the effect of nucleoside analogue resistance mutations on the efficiency and kinetics of HIV-1 DNA synthesis and to evaluate the impact of the depletion of deoxynucleoside triphosphates (dNTP) on this process, DNA synthesis was evaluated by allowing DNA synthesis to proceed with natural HIV-1 templates and primers, either within permeabilized viral particles or in newly infected cells, and quantifying the products by real-time PCR. Three recombinant viruses derived from three pNL4-3 molecular clones expressing mutations associated with resistance to zidovudine: a clone expressing RT mutation M184V, a clone expressing mutations M41L plus T215Y (M41L+T215Y), and clinical isolate BV34 (carrying seven resistance mutations). Following infection of P4 cells, the BV34 mutant, but not viruses expressing the M184V mutation or M41L+T215Y, exhibited a defect in DNA synthesis. Importantly, however, for mutants carrying the M184V mutation or M41L+T215Y mutations, a defect could be detected by using target cells in which dATP pools had been reduced by pretreatment with hydroxyurea. Based on these observations, we developed a recombinant-virus assay to assess the effects of hydroxyurea pretreatment on infectivity of viruses carrying plasma-derived RT sequences from patients with nucleoside resistance. Using this assay, we found that many, but not all, viruses carrying RT resistance mutations display an increased sensitivity to hydroxyurea, suggesting that the impact of RT resistance mutations on viral replication may be more profound in cell populations characterized by smaller dNTP pools.

* Corresponding author. Mailing address: INSERM U.552, Hôpital Bichat-Claude Bernard, 46, rue Henri Huchard, 750918 Paris, France. Phone: 33-1-40-25-63-55. Fax: 33-1-40-25-63-70. E-mail: hance{at}bichat.inserm.fr.

Journal of Virology, January 2005, p. 812-822, Vol. 79, No. 2
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.2.812-822.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Perez-Bercoff, D., Wurtzer, S., Compain, S., Benech, H., Clavel, F. (2007). Human Immunodeficiency Virus Type 1: Resistance to Nucleoside Analogues and Replicative Capacity in Primary Human Macrophages. J. Virol. 81: 4540-4550 [Abstract] [Full Text]  
  • Wurtzer, S., Goubard, A., Mammano, F., Saragosti, S., Lecossier, D., Hance, A. J., Clavel, F. (2006). Functional Central Polypurine Tract Provides Downstream Protection of the Human Immunodeficiency Virus Type 1 Genome from Editing by APOBEC3G and APOBEC3B.. J. Virol. 80: 3679-3683 [Abstract] [Full Text]  
  • Wurtzer, S., Compain, S., Benech, H., Hance, A. J., Clavel, F. (2005). Effect of Cell Cycle Arrest on the Activity of Nucleoside Analogues against Human Immunodeficiency Virus Type 1. J. Virol. 79: 14815-14821 [Abstract] [Full Text]  


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