16th International HIV Drug Resistance Workshop 12-16 June 2007, Barbados

NOVEL PATHWAYS TO NCRTI RESISTANCE SELECTED IN VITRO FROM HIV-1 STRAINS

Antivir Ther. 2007; 12:S25 (abstract no. 23)

D Jochmans, M Van Ginderen, I De Baere, S Hallenberger and G Kraus
Tibotec, Mechelen, Belgium

BACKGROUND: Nucleotide-competing reverse transcriptase inhibitors (NcRTIs) are a novel class of RT inhibitors. Previous in vitro selection (IVS) experiments showed two independent pathways towards NcRTI resistance: selection of M184V together with other mutations, and selection of L234F. Additionally, we demonstrated that K65R is associated with hypersensitivity for the prototype compound NcRTI-1. We performed further IVS experiments to study NcRTI resistance pathways. In addition, an automated 96-well plate IVS method was evaluated.

METHODS: Classical IVS method: Virus replication was initiated by infecting MT4 cells at MOI=0.01 and 0.1 µM NcRTI-1 (~3×EC₅₀). Upon full cytopathic effect, harvested viruses were used to re-infect new MT4 cell cultures containing a three times increased concentration of NcRTI-1. Automated 96-well plate IVS: MT4 cells were infected at MOI=0.01 in the presence of different NcRTI-1 concentrations (0.3–300×EC₅₀). Every 3–4 days, supernatant from each culture was used to re-infect three new cultures with four, two and one times increased NcRTI concentration.

RESULTS: Independent classical IVS experiments were started from HIV-1 IIIB (n=14), NL4.3 (n=7) and recombinant clinical isolates (n=6). In addition 28 viruses were selected from NL4.3 using the automated method. Population genotyping of the selected viruses showed the existence of four independent pathways towards NcRTI resistance, occurring irrespective of the input strain or the IVS method used. The major pathway (55% of experiments) was the selection of M184V together with additional mutations. The other pathways were the selection of A62V+A114S (20%), V111G (15%) and L234F (10%). The association of M184V, A62V+A114S and L234F with NcRTI-1 resistance was confirmed in antiviral testing. The V111G mutant could not be propagated for further analysis. Infrequently we observed the selection of Y181C during IVS from IIIB. However, since Y181C was never selected in experiments starting from NL4.3 (n=35) and because it did not significantly affect the susceptibility for NcRTI-1 (FC~2×), the relevance of this observation is unclear, and needs further investigation.

CONCLUSION: Different IVS methods reveal novel NcRTI resistance pathways via mutations in the active site of RT (A62V+A114S and V111G). This strengthens the hypothesis that NcRTIs inhibit HIV-1 RT by binding the active site and competing with incoming nucleotides.

2007-06-12
23

Copyright © 2006 - International Medical Press Ltd.. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the International Medical Press Ltd. 2-4 Idol Lane, London EC3R 5DD UK.