Springer Protocols: Abstract: In Vitro Translation to Study HIV Protease Activity

In Vitro Translation to Study HIV Protease Activity

By: Zene Matsuda², Mutsunori Iga³, Kosuke Miyauchi⁴, Jun Komano⁵, Kazuhiro Morishita^{6 7}, Akihiko Okayama⁸, Hirohito Tsubouchi⁹

Abstract

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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.

Affiliation(s): (2) Research Center for Asian Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
(3) Division of Infectious Diseases, The Advanced Clinical Research Center, The Institute of Medical Science, The Unviersity of Tokyo, Tokyo, Japan
(4) Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, MD
(5) Laboratory of Virology and Pathogenesis, AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan
(6) Department of Tumor and Cellular Biochemistry, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
(7) Department of Biochemistry, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
(8) Department of Rheumatology, Infectious Diseases, and Laboratory Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
(9) Department of Digestive and Life-Style Related Disease, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

Book Title: In Vitro Transcription and Translation Protocols

Series: Methods in Molecular Biology | Volume: 375 | Pub. Date: May-03-2007 | Page Range: 135-149 | DOI: 10.1007/978-1-59745-388-2_7

Subject: Genetics/Genomics

Key Words: HIV-1 - protease - genotyping - phenotyping - drug resistance - ELISA - protease inhibitor - p24

References

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