Mutations in the non-nucleoside binding-pocket interfere with the multi-nucleoside resistance phenotype

Objectives: To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucl eoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV -1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. Methods: Virus strains were selected in C8166 cells in the presence of incr easing concentrations of lamivudine or HBY097. In parallel control experime nts, the virus was cultured in C8166 cells in the absence of drugs. The ent ire reverse transcriptase encoding region was amplified using polymerase ch ain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. Results: High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleosid e resistance mutations were stable during in vitro resistance selection. La mivudine elicited the acquisition of the M1841 mutation. Phenotypic resista nce to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was i ncreased when M1841 was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resi stance selection, at least two mutations associated with NNRTI resistance r esulted in high-level NNRTI resistance. The NNRTI resistance-related mutati ons partially reversed the phenotypic resistance to most NRTIs, except to a bacavir. The addition of the M1841 mutation to the NNRTI-multi-nucleoside r esistance set abolished this antagonizing effect for didanosine, zalcitabin e and lamivudine, but further potentiated the phenotypic reversal for zidov udine and stavudine. Conclusion: Changes in the non-nucleoside binding pocket must affect the co nformation of residues at the dNTP binding site, and can result in a partia l phenotypic reversal of the multi-nucleoside resistance phenotype. (C) 200 1 Lippincott Williams & Wilkins.