3rd International Workshop on HIV Drug Resistance 2-5 August 1994, Kauai, Hawaii, USA

REDUCED IN VITRO POLYMERASE ACTIVITY OF 3TC-RESISTANT REVERSE TRANSCRIPTASE ENZYMES

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:32 (abstract no. 31)

Nicole K.T. Back, Monique Nijhuis, Charles A.B. Boucher, Dorien de Jong, Rob Schuurman, Belinda Oude Essink, and Ben Berkhout
Department of Virology, University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands

During the first 12 weeks of 3TC therapy, viruses carrying 3TC resistance mutations appear. Initially, codon 184 Isoleucine virus variants (M 184I) are detectable, followed by the appearance and eventual outgrowth of viruses encoding an 184 Valine (M184V). The aim of this study was to investigate the replication potential of the wild type.virus (Methionine at position 184) and these 3TC-resistant variants since amino acid 184 is part of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. The 184 mutants were constructed by site-directed mutagenesis in the HXB2 background. Viruses were generated by co-electroporation of an RT-deleted HXB2 molecular clone and the different RT gene fragments into the SupT1 T-cell line. Virus stocks were harvested as soon as full-blown syncytia had developed and were titrated on SupT1 cells to determine their TCID₅₀. No differences in viral replication kinetics were measured for wild type and 3TC-resistant viruses ba~ on Gag p24 assays.

Next, we measured virion-associated RT activity on poly (rA): oligo (dT) in vitro. Interestingly, we consistently measured reduced RT activity for both 3TC-resistant RT variants compared to the wild type enzyme. We measured 35-59% and 43-70% activity for the M184I and M184V variants, respectively. Gel analysis of the poly (dT) products showed that both mutant RT enzymes produce significantly shorter cDNA products than the wild type enzyme.

These results suggest that 3TC-resistant RT is less processive in vitro. The M184V RT protein was slightly more active in the polyA: dT assay compared to the M184I variant, consistent with the in vivo outgrowth of the M184V virus.

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1994-08-02
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