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In Vitro Translation to Study HIV Protease Activity

  • Zene Matsuda
  • Mutsunori Iga
  • Kosuke Miyauchi
  • Jun Komano
  • Kazuhiro Morishita
  • Akihiko Okayama
  • Hirohito Tsubouchi
Part of the Methods in Molecular Biology™ book series (MIMB, volume 375)

Summary

HIV-1 is an etiological agent of AIDS. One of the targets of the current anti-HIV-1 combination chemotherapy, called highly active antiretroviral therapy (HAART), is HIV-1 protease (PR), which is responsible for the processing of viral structural proteins and, therefore, essential for virus replication. Here, we describe an in vitro transcription/translation-based method of phenotyping HIV-1 PR. In this system, both substrate and PR for the assay can be prepared by in vitro transcription/translation. Protease activity is estimated by the cleavage of a substrate, as measured by enzyme-linked immunosorbent assay (ELISA). This assay is safe, rapid, and requires no special facility to be carried out. Our rapid phenotyping method of HIV-1 PR may help evaluate drug resistance, useful when choosing an appropriate therapeutic regiment, and could potentially facilitate the discovery of new drugs effective against HIV-1 PR.

Key Words

HIV-1 protease genotyping phenotyping drug resistance ELISA protease inhibitor p24 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Zene Matsuda
    • 1
  • Mutsunori Iga
    • 2
  • Kosuke Miyauchi
    • 3
  • Jun Komano
    • 4
  • Kazuhiro Morishita
    • 5
    • 6
  • Akihiko Okayama
    • 7
  • Hirohito Tsubouchi
    • 8
  1. 1.Research Center for Asian Infectious Diseases, The Institute of Medical ScienceThe University of TokyoTokyoJapan
  2. 2.Division of Infectious Diseases, The Advanced Clinical Research Center, The Institute of Medical ScienceThe Unviersity of TokyoTokyoJapan
  3. 3.Institute of Human VirologyUniversity of Maryland Biotechnology InstituteBaltimore
  4. 4.Laboratory of Virology and Pathogenesis, AIDS Research CenterNational Institute of Infectious DiseasesTokyoJapan
  5. 5.Department of Tumor and Cellular Biochemistry, Faculty of MedicineUniversity of MiyazakiMiyazakiJapan
  6. 6.Department of Biochemistry, Faculty of MedicineUniversity of MiyazakiMiyazakiJapan
  7. 7.Department of Rheumatology, Infectious Diseases, and Laboratory Medicine, Faculty of MedicineUniversity of MiyazakiMiyazakiJapan
  8. 8.Department of Digestive and Life-Style Related DiseaseKagoshima University Graduate School of Medical and Dental SciencesKagoshimaJapan

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