4th International Workshop on HIV Drug Resistance & Treatment Strategies Sitges, Spain, 12–16 June 2000

BIOCHEMICAL MECHANISM OF HIV-1 REVERSE TRANSCIPTASE RESISTANCE TO STAVUDINE

Antivir Ther. 2000 Jun 12-16; 5 (Suppl. 3):14 (Abstract 16

J Lennerstrand¹, K Hertogs², DK Stammers³ and B Larder^1
1Virco, Cambridge, UK; ²Virco, Mechelen, Belgium; and ³Wellcome Trust Centre for Human Genetics, Oxford University, UK

OBJECTIVE: The mechanisms of stavudine resistance for HIV-1 are not well known. To investigate these mechanisms we have constructed site-directed mutants that are known, or suspected to confer reduced stavudine susceptibility. Constructs chosen for assays were the V75T mutation alone, codon-69 insertions (T69SSG and -AG) alone and combined with zidovudine resistance mutations.

METHODS: The inhibitor susceptibility of these mutants relative to wild-type (HXB2) was studied by (i) recombinant virus assay, and (ii) a non-radioactive reverse transcriptase (RT) assay (prA/odT system) performed with stavudine-TP with and without added physiological concentration of ATP.

RESULTS: The highest level of stavudine resistance (17-fold) was shown in the recombinant virus assay by viruses containing T69S-AG together with mutations M41L, L210W, R211K, L214F and T215Y. The single site T69S-AG and -SG mutants showed a seven- and a four-fold reduced sensitivity, respectively. However, virus with the single site V75T only exhibited a three-fold reduced sensitivity. In the RT assay with addition of ATP, taking the K_m(BrdUTP) into account together with the K_i(stavudine-TP), the T69S-AG alone and together with M41L, L210W, R211K, L214F and T215Y showed a five- and 10-fold decreased sensitivity for stavudine-TP, respectively. When conducting the RT assay without ATP, the 69 insertion mutants behaved as wild-type enzyme. However, the V75T mutant, despite its moderate phenotypic resistance level, showed the same three-fold decreased sensitivity in the RT assay both with and without added ATP.

CONCLUSIONS: These results indicate that the 69 insertion mutations we studied could be involved in the same ATP-dependent mechanism analogous to pyrophosphorolysis as we previously reported for resistance to zidovudine-TP at the enzyme level. In contrast, the V75T mutant RT seems to be involved in decreased binding to stavudine-TP independently of added ATP.

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2000-06-12
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