title: Construction of a human immunodeficiency virus type 1 (HIV-1) library containing random combinations of amino acid substitutions in the HIV-1 protease due to resistance by protease inhibitors.
abstract: Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 x 10(5)) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 microM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.
authors.affiliation: Department of Medical Virology, Kumamoto University School of Medicine, Kumamoto University, Kumamoto 860-0811, Japan. yusak@kaiju.medic.kumamoto-u.ac.jp
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yusa
authors.forename: Wei
authors.initials: W
authors.lastname: Song
authors.forename: Matthias
authors.initials: M
authors.lastname: Bartelmann
authors.forename: Shinji
authors.initials: S
authors.lastname: Harada
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002460:Cell Line; D024882:Drug Resistance, Viral; D015723:Gene Library; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D019438:Ritonavir / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000821:virology
chemical_list: D017320:HIV Protease Inhibitors; D016333:HIV Protease; D019438:Ritonavir