title: Novel mutations in reverse transcriptase of human immunodeficiency virus type 1 reduce susceptibility to foscarnet in laboratory and clinical isolates.
abstract: Foscarnet (phosphonoformic acid) is a pyrophosphate analog that inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in vitro and in patients with AIDS. HIV-1 resistance to foscarnet has not been reported despite long-term foscarnet therapy of AIDS patients with cytomegalovirus disease. We therefore attempted to select foscarnet-resistant HIV-1 in vitro by serial endpoint passage of virus in 400 microM foscarnet. After 13 cycles of passage in MT-2 cells, virus exhibiting > or = 8.5-fold foscarnet resistance was isolated. The reverse transcriptase (RT) from resistant virions exhibited a similar level of foscarnet resistance in enzyme inhibition assays (approximately 10-fold resistance). Foscarnet-resistant virus showed increased susceptibility to 3'-azido-3'-deoxythymidine (90-fold) and to the HIV-1-specific RT inhibitors TIBO R82150 (30-fold) and nevirapine (20-fold). DNA sequence analysis of RT clones from resistant virus revealed the coexistence of two mutations in all clones: Gln-161 to Leu (CAA to CTA) and His-208 to Tyr (CAT to TAT). Sequence analysis of six clinical HIV-1 isolates showing reduced susceptibility to foscarnet revealed the Tyr-208 mutation in two, the Leu-161 mutation in one, and a Trp-88-to-Ser or -Gly mutation in four isolates. Site-specific mutagenesis and production of mutant recombinant viruses demonstrated that the Leu-161, Ser-88, and Tyr-208 mutations reduced HIV-1 susceptibility to foscarnet 10.5-, 4.3-, and 2.4-fold, respectively, in MT-2 cells. In the crystal structure of HIV-1 RT, the Gln-161 residue lies in the alpha E helix beneath the putative deoxynucleoside triphosphate (dNTP) binding site. The Gln-161-to-Leu mutation may affect the structure of the dNTP binding site and its affinity for foscarnet. The location of the Trp-88 residue in the Beta5a strand of HIV-1 RT suggest that the Ser-88 mutation affects template-primer binding, as do several mutations that affect RT susceptibility to nucleoside analogs.
authors.affiliation: Department of Medicine, University of Pittsburg School of Medicine, PA 15261, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Mellors
authors.forename: H Z
authors.initials: HZ
authors.lastname: Bazmi
authors.forename: R F
authors.initials: RF
authors.lastname: Schinazi
authors.forename: B M
authors.initials: BM
authors.lastname: Roy
authors.forename: Y
authors.initials: Y
authors.lastname: Hsiou
authors.forename: E
authors.initials: E
authors.lastname: Arnold
authors.forename: J
authors.initials: J
authors.lastname: Weir
authors.forename: D L
authors.initials: DL
authors.lastname: Mayers
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis; D017245:Foscarnet / Q000494:pharmacology*; D015658:HIV Infections / Q000201:enzymology / Q000821:virology*; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D018894:Reverse Transcriptase Inhibitors
chemical_list: D004279:DNA, Viral; D018894:Reverse Transcriptase Inhibitors; D017245:Foscarnet; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase