REDUCED REPLICATION OF 3TC-RESISTANT HIV-1 VARIANTS IN PRIMARY-CELLS DUE TO A PROCESSIVITY DEFECT OF THE REVERSE-TRANSCRIPTASE ENZYME

Human immunodeficiency virus type 1 (HIV-1) variants with resistance m utations in the reverse transcriptase (RT) gene appear during drug the rapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved Y (M) under bar DD motif, which is part of the catalytic cor e of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventually outgrowth of viruses encodin g valine (Val). Similar replication kinetics were measured for wild-ty pe and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mu tant RT enzymes compared with the wild-type enzyme (Ile 43% and Val = 67%). Gel analysis of the reverse transcription products revealed that both 3TC-resistant RT mutants produce significantly shorter cDNA mole cules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3T C-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concent rations and we therefore assayed virus replication in primary cells th at contain relatively low dNTP levels. Under these conditions, we meas ured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication c an be similarly reduced in 3TC-treated patients that develop drug-resi stant HIV-1 variants, this may be of considerable clinical benefit.