1st International Workshop on HIV Drug Resistance & Treatment Strategies 25-28 June 1997, St. Petersburg, Florida, USA

THE INFLUENCE OF INCREASED REVERSE TRANSCRIPTASE FIDELITY OF CODON 184 VARIANTS USING COMPETITION EXPERIMENTS UNDER ANTIVIRAL PRESSURE

Antiviral Therapy 1997;2 (Suppl 5):22 (abstract no. 34)

Wilco Keulen¹, Monique Nijhuis¹, Albert van Wijk¹, Rob Schuurman¹, Ben Berkhout² and Charles Boucher^1
1Department of Virology, Eijkman Winkler Institute, University Hospital Utrecht; and ²Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, The Netherlands.

Since the observation that the reverse transcriptase (RT) enzyme of the lamivudine resistant-RT variant (184Val) showed an enhanced fidelity in nucleotide insertion experiments, many researchers have performed in vitro selection experiments to investigate the effect of enhanced fidelity on the generation of resistance. Last year we presented data showing that the increase in RT fidelity was not able to prevent the generation of double resistant viruses [1]. However with this type of in vitro selections small differences in the kinetics of the appearance of double resistant variants are difficult to detect. Therefore we used a novel approach in which mixtures of wild-type and codon 184 mutant viruses were placed under antiviral pressure (nevirapine or ritonavir). Subsequently, differences in the capacity to acquire additional mutations was monitored by the outgrowth of one of the variants.

The experiments were performed in supT1 cells with either mixtures of wild-type 184Met with 184Ile variant (M/I mixture), M/V mixture or I/V mixture. After five passages with increasing drug concentrations, the virus population of each selection was analysed by direct sequencing. The wild-type virus was only able to outgrow the lamivudine resistant variant in the M/V mixture with nevirapine as selective pressure. In the other selections the wild-type virus was out-competed by the mutant 184 variants. In both I/V mixtures, the virus harbouring the 184Ile codon was able to outgrow the 184Val variant.

In conclusion, with this novel approach we observe differences in the capacity to generate drug-resistant mutations between the 184 variants. However the outgrowth of the codon 184 variants was also observed in control competition experiments in the absence of drug. This indicates that the overall fitness of the codon 184 mutant variants is higher in supT1 cells than wild-type virus and that other factors than the capacity to generate secondary resistant mutations are responsible of the outgrowth of the lamivudine resistant variants.

1. Keulen W, Nijhuis M, Schuurman R, Berkhout B & Boucher C. The observed increase in enzyme fidelity of the 184Val variant is not sufficient to prevent the generation of double resistant HIV-1 viruses. Antiviral Therapy 1996; 1(Supplement 1): Abstract 11.

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1997-06-25
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