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.
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.
Koh
Yasuhiro
Y
Department of Infectious Diseases, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan.
Amano
Masayuki
M
Towata
Tomomi
T
Danish
Matthew
M
Leshchenko-Yashchuk
Sofiya
S
Das
Debananda
D
Nakayama
Maki
M
Tojo
Yasushi
Y
Ghosh
Arun K
AK
Mitsuya
Hiroaki
H
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*
D017320:HIV Protease Inhibitors; D014764:Viral Proteins; D019438:Ritonavir
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