title: Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.
abstract: Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val). Similar replication kinetics were measured for wild-type and 3TC-resistant HIV-1 viruses in tissue culture infections of a T cell line, but we measured reduced polymerase activity for the two mutant 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 molecules than the wild-type enzyme [Met (wt)>Val>Ile], indicating that 3TC-resistant RT polymerases are less processive enzymes. Interestingly, these enzyme defects were more pronounced under limiting dNTP concentrations and we therefore assayed virus replication in primary cells that contain relatively low dNTP levels. Under these conditions, we measured significantly reduced replication kinetics for the 3TC-resistant HIV-1 variants [Met (wt)>Val>Ile]. If the level of virus replication can be similarly reduced in 3TC-treated patients that develop drug-resistant HIV-1 variants, this may be of considerable clinical benefit.
authors.affiliation: Department of Human Retrovirology, University of Amsterdam, Academic Medical Center, Utrecht, Netherlands.
authors.forename: N K
authors.initials: NK
authors.lastname: Back
authors.forename: M
authors.initials: M
authors.lastname: Nijhuis
authors.forename: W
authors.initials: W
authors.lastname: Keulen
authors.forename: C A
authors.initials: CA
authors.lastname: Boucher
authors.forename: B O
authors.initials: BO
authors.lastname: Oude Essink
authors.forename: A B
authors.initials: AB
authors.lastname: van Kuilenburg
authors.forename: A H
authors.initials: AH
authors.lastname: van Gennip
authors.forename: B
authors.initials: B
authors.lastname: Berkhout
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D004261:DNA Replication; D003853:Deoxyribonucleosides / Q000378:metabolism; D004352:Drug Resistance, Microbial; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D019259:Lamivudine; D015636:Magnesium Chloride / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects*; D016047:Zalcitabine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D003853:Deoxyribonucleosides; D018894:Reverse Transcriptase Inhibitors; D015636:Magnesium Chloride; D019259:Lamivudine; D016047:Zalcitabine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase