Rapid phenotypic assay for human immunodeficiency virus type 1 protease using in vitro translation

Mutsunori Iga, Zene Matsuda, Akihiko Okayama, Wataru Sugiura, Seiichi Hashida, Kazuhiro Morishita, Yoshiyuki Nagai, Hirohito Tsubouchi

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

A rapid in vitro phenotyping method for human immunodeficiency virus type 1 (HIV-1) protease was developed. In this system, both HIV-1 protease and substrates are prepared using a rabbit reticulocyte based coupled in vitro transcription/translation system. The activity of protease is evaluated by the amount of cleaved substrate measured by ELISA. In this system, wild-type protease derived from strain HXB2 was specifically inhibited in a dose-dependent manner by the protease inhibitors, indinavir and nelfinavir. Three drug-resistant proteases carrying a single mutation, D30N, L90M, and V82F, were analyzed in the absence of the inhibitors. Reflecting their impaired fitness, they exhibited decreased protease activity compared with the wild type. The apparent protease activity was greater for a Gag-Pol substrate encompassing the Gag-protease-reverse transcriptase junctions than for a substrate only covering the Gag region. Using the Gag-Pol substrate as the target, the indinavir-resistant mutant V82F was evaluated further. V82F showed 9-fold resistance to its cognitive protease inhibitor, indinavir; however, it manifested only moderate (2-fold) resistance to a non-cognitive inhibitor, nelfinavir. This simple and rapid method may be useful for phenotyping of drug-resistant HIV-1 protease as well as for screening new inhibitors of HIV-1 protease.

Original languageEnglish
Pages (from-to)25-37
Number of pages13
JournalJournal of Virological Methods
Volume106
Issue number1
DOIs
Publication statusPublished - Oct 1 2002

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HIV-1
Peptide Hydrolases
Indinavir
Nelfinavir
Protease Inhibitors
In Vitro Techniques
Reticulocytes
RNA-Directed DNA Polymerase
Pharmaceutical Preparations
Enzyme-Linked Immunosorbent Assay
Rabbits
Mutation

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

Rapid phenotypic assay for human immunodeficiency virus type 1 protease using in vitro translation. / Iga, Mutsunori; Matsuda, Zene; Okayama, Akihiko; Sugiura, Wataru; Hashida, Seiichi; Morishita, Kazuhiro; Nagai, Yoshiyuki; Tsubouchi, Hirohito.

In: Journal of Virological Methods, Vol. 106, No. 1, 01.10.2002, p. 25-37.

Research output: Contribution to journalArticle

Iga, M, Matsuda, Z, Okayama, A, Sugiura, W, Hashida, S, Morishita, K, Nagai, Y & Tsubouchi, H 2002, 'Rapid phenotypic assay for human immunodeficiency virus type 1 protease using in vitro translation', Journal of Virological Methods, vol. 106, no. 1, pp. 25-37. https://doi.org/10.1016/S0166-0934(02)00133-7
Iga, Mutsunori ; Matsuda, Zene ; Okayama, Akihiko ; Sugiura, Wataru ; Hashida, Seiichi ; Morishita, Kazuhiro ; Nagai, Yoshiyuki ; Tsubouchi, Hirohito. / Rapid phenotypic assay for human immunodeficiency virus type 1 protease using in vitro translation. In: Journal of Virological Methods. 2002 ; Vol. 106, No. 1. pp. 25-37.
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