15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

STRONG DECREASE IN VIRAL REPLICATION CAPACITY IN HIV-1 CONTAINING BOTH K65R AND Q151M MUTATIONS

Antivir Ther. 2006, 11:S44 (abstract no. 36)

J Garcia-Perez¹, M Perez-Olmeda¹, S Sanchez-Palomino², L Menendez-Arias³, L Valer⁴, F Garcia⁵, T Pumarola², V Soriano⁴ and J Alcami^1
1Instituto de Salud Carlos III, Madrid, Spain; ²Hospital Clínic, IDIBAPS, University of Barcelona, Spain; ³Centro de Biologia Molecular “Severo Ochoa”, CSIC–UAM, Madrid, Spain; ⁴Hospital Carlos III, Madrid, Spain; ⁵Hospital San Cecilio, Granada, Spain

BACKGROUND: An inversely correlation between drug resistance mutations and viral replication capacity (RC) has been often described in HIV infection. Interestingly K65R is frequently associated with the Q151M mutation but the impact of this combination on HIV-RC has not been assessed so far. Using different “in vitro” methods we have determined the impact of different combinations of K65R, Q151M complex (Q151Mc) and M184V mutations on viral fitness.

METHODS: We have generated a NL 4.3-based viral clone containing the Renilla-Luciferase gene as reporter that is able to produce multiple cycles of HIV-replication in culture. This system allows the analysis of RC of viral populations in which only RT sequences from patients are cloned. RC of viral populations from 4 WT and 49 HIV-patients in therapeutic failure with different combinations of K65R, M184V and Q151Mc mutations were analysed. RT sequences from plasma were amplified, sequenced and used for generation of RV. All the combinations of mutations were also generated by site-directed mutagenesis. Viral progeny was obtained through transfection in 293-T cells, normalized by luciferase activity and used to infect MT-2 cells. Relative RC was assessed by comparing luciferase activity between mutant RV and WT clinical isolates or the reference virus.

Replication kinetics assays and growth competition experiments with site-directed mutants were done in MT-2 at low multiplicity of infection. The catalytic efficiency of these viral enzymes were also analysed by primer extension and pre-steady state kinetic assays.

RESULTS: The analysis of relative RC in comparison with WT virus was 69% (K65R), 95% (Q151Mc), 73% (M184V), 40% (K65R+M184V), 42% (K65R+Q151Mc), 74% (Q151M+M184V) and 27% (K65R+Q151Mc+M184V). When patient-derived RVs were analysed a similar pattern of replication was found. Replication kinetics assays and growth competition experiments confirmed that viruses containing the combination K65R+Q151Mc showed stronger decrease in their RC as compared with single mutants. The catalytic efficiency (k_pol/k_d ) for WT, K65R, Q151Mc and K65R+Q151Mc enzymes were 1.28, 0.90, 1.06 and 0.75 s^-1 µM^-1 respectively.

CONCLUSIONS: A strong decrease (60%) in viral RC was observed in RV containing the K65R in association with Q151Mc as compared with single K65R (30%) and Q151Mc (5–10%) mutations.

2006-06-13
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