title: In vitro selection of highly darunavir-resistant and replication-competent HIV-1 variants by using a mixture of clinical HIV-1 isolates resistant to multiple conventional protease inhibitors.
abstract: We attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has a high genetic barrier to HIV-1 development of resistance to DRV. We conducted selection using a mixture of 8 highly multi-protease inhibitor (PI)-resistant, DRV-susceptible clinical HIV-1 variants (HIV-1(MIX)) containing 9 to 14 PI resistance-associated amino acid substitutions in protease. HIV-1(MIX) became highly resistant to DRV, with a 50% effective concentration (EC(50)) ∼333-fold greater than that against HIV-1(NL4-3). HIV-1(MIX) at passage 51 (HIV-1(MIX(P51))) replicated well in the presence of 5 μM DRV and contained 14 mutations. HIV-1(MIX(P51)) was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1(MIX(P51)) had a resemblance with HIV-1(C) of the HIV-1(MIX) population, and selection using HIV-1(C) was also performed; however, its DRV resistance acquisition was substantially delayed. The H219Q and I223V substitutions in Gag, lacking in HIV-1(C(P51)), likely contributed to conferring a replication advantage on HIV-1(MIX(P51)) by reducing intravirion cyclophilin A content. HIV-1(MIX(P51)) apparently acquired the substitutions from another HIV-1 strain(s) of HIV-1(MIX) through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance; however, HIV-1 can develop high levels of DRV resistance when a variety of PI-resistant HIV-1 strains are generated, as seen in patients experiencing sequential PI failure, and ensuing homologous recombination takes place. HIV-1(MIX(P51)) should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents.
authors.affiliation: Department of Infectious Diseases, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Koh
authors.forename: Masayuki
authors.initials: M
authors.lastname: Amano
authors.forename: Tomomi
authors.initials: T
authors.lastname: Towata
authors.forename: Matthew
authors.initials: M
authors.lastname: Danish
authors.forename: Sofiya
authors.initials: S
authors.lastname: Leshchenko-Yashchuk
authors.forename: Debananda
authors.initials: D
authors.lastname: Das
authors.forename: Maki
authors.initials: M
authors.lastname: Nakayama
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Tojo
authors.forename: Arun K
authors.initials: AK
authors.lastname: Ghosh
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019438:Ritonavir / Q000494:pharmacology*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D017320:HIV Protease Inhibitors; D014764:Viral Proteins; D019438:Ritonavir
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