13th International HIV Drug Resistance Workshop


8–12 June 2004, Tenerife Sur-Costa Adeje, Canary Islands, Spain


EFFECTS OF MUTATIONS ASSOCIATED WITH SUPPRESSION OF ZIDOVUDINE RESISTANCE IN HIV-1 REVERSE TRANSCRIPTASE ON REMOVAL OF TENOFOVIR FROM BLOCKED PRIMER/TEMPLATE

Antiviral Therapy 2004; 9:S28

N Hassani Espili1, T Bergroth1, A Pavlova1, X-W Shao2, P Mc Kenna3, A Sönnerborg1, J Lennerstrand1
1Karolinska Institute, Huddinge University Hospital, Stockholm; 2Karolinska Institute, MTC, Stockholm, Sweden; and 3Virco NV, Mechelen, Belgium

BACKGROUND: It has been reported that the M184V mutation in HIV-1 reverse transcriptase (RT) interferes with ATP mediated excision of zidovudine (AZT)-MP. However, depending on the type of RT assay used, there have been contradictory results regarding the effects of the M184V mutation on excision. We have now investigated the effect of known AZT suppression mutations (M184V and Y181C) for tenofovir unblocking, in a background of multi-nucleoside resistance mutations.

METHODS: Site directed RT mutants were constructed carrying: M41L, D67N, K70R, L210W, T215Y (TAM mutant) and M41L, T69S-SG, L210W, T215Y (69SSG+ TAM mutant). The M184V mutation was added to wild-type, TAM mutant and the 69S-SG+TAM mutant, respectively. Also, Y181C was added to the 69SSG+ TAM mutant. Tenofovir resistance was studied by recombinant virus assay (Antivirogram®, Virco, Belgium). The ATP mediated excision of tenofovir was studied for the RT mutants, purified in the form of p66/p51 heterodimers, with a non-radioactive RT assay (Cavidi Tech, Sweden), using a long hetero-polymeric DNA template and low concentrations of dNTP.

RESULTS: The mutants carrying the 69S-SG insertion were analysed with the recombinant virus assay. The relative increase in IC50 for tenofovir compared to wild-type virus was: 69S-SG+TAM, 17.8; 69SSG+ TAM+184V, 3.2; and 69S-SG+TAM+181C, 7.5. In studies of purified RT mutants in the RT assay with ATP added, the level of tenofovir-DP IC50 relative to wild type RT were as follows: 184V, 0.5; TAM, 2.8; TAM+184V, 1.5; 69S-SG+TAM, 10.2; 69S-SG +TAM+184V, 2.8; and 69S-SG+TAM+181C, 2.5. When conducting the RT assay without ATP, the mutants behaved similar to wild-type enzyme (1 ±0.4), with exception for 69S-SG+TAM, which showed a non-ATP dependent 2.6 times decreased tenofovir incorporation rate. Furthermore, inhibition by dNTP complementary to the next nucleotide position on primer blocked tenofovir and ddA-MP template was studied for the 69SG+TAM mutant. Removal of tenofovir was found to be somewhat less sensitive to inhibition by the next dNTP than was removal of ddA-MP.

CONCLUSIONS: The M184V mutation seems to reduce the ATP mediated excision of incorporated tenofovir both in a background of wild-type and multiple thymidine analogue-associated mutations. Furthermore, significant suppression of tenofovir resistance was found for both M184V and Y181C in the presence of T69S-SG insertion together with thymidine analogue-associated mutations. Thus, our biochemical data is consistent with cell culture data. This indicates that there would possibly be a benefit with tenofovir therapy, combined with therapy rendering suppression mutations, and these findings should therefore be taken into account in genotyping interpretations.

PRESENTING AUTHOR: J Lennerstrand

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2004-06-08
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