title: Effects of zidovudine-selected human immunodeficiency virus type 1 reverse transcriptase amino acid substitutions on processive DNA synthesis and viral replication.
abstract: Certain amino acid substitutions in the reverse transcriptase (RT), including D67N, K70R, T215Y, and K219Q, cause high-level resistance of human immunodeficiency virus type 1 (HIV-1) to zidovudine (3'-azidothymidine; AZT) and appear to approximate the template strand of the enzyme-template-primer complex in structural models. We studied whether this set of mutations altered RT-template-primer interaction as well as their effect on virus replication in the absence of inhibitor. When in vitro polymerization was limited to a single association of an RT with an oligodeoxynucleotide-primed heteropolymeric RNA template (a single processive cycle), recombinant-expressed mutant 67/70/215/219 RT synthesized 5- to 10-fold more high-molecular-weight DNA products (>200 nucleotides in length) than wild-type RT. This advantage was maintained as deoxynucleoside triphosphate (dNTP) concentrations were decreased to limiting levels. In contrast, no difference was seen between wild-type and mutant RTs under conditions allowing repeated associations of enzyme with template-primer. Because intracellular dNTP concentrations are low prior to mitogenic stimulation, we compared replication of mutant 67/70/215/219 virus and wild-type virus in peripheral blood mononuclear cells (PBMC) stimulated before and after infection. In the absence of inhibitor, mutant 67/70/215/219 virus had a replication advantage in PBMC stimulated with phytohemagglutinin and interleukin-2 after infection, but virus replication was similar in PBMC stimulated before infection in vitro. The results confirm that RT mutations D67N, K70R, T215Y, and K219Q affect an enzyme-template-primer interaction in vitro and suggest that such substitutions may affect HIV-1 pathogenesis during therapy by increasing viral replication capacity in cells stimulated after infection.
authors.affiliation: Infectious Disease Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Caliendo
authors.forename: A
authors.initials: A
authors.lastname: Savara
authors.forename: D
authors.initials: D
authors.lastname: An
authors.forename: K
authors.initials: K
authors.lastname: DeVore
authors.forename: J C
authors.initials: JC
authors.lastname: Kaplan
authors.forename: R T
authors.initials: RT
authors.lastname: D'Aquila
mesh_terms: D001483:Base Sequence; D001704:Biopolymers; D002478:Cells, Cultured; D004279:DNA, Viral / Q000096:biosynthesis / Q000187:drug effects; D003854:Deoxyribonucleotides / Q000378:metabolism; D004352:Drug Resistance, Microbial; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects*; D015215:Zidovudine / Q000494:pharmacology*
chemical_list: D001704:Biopolymers; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D015215:Zidovudine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase