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  • Title: Establishment of an in vitro assay to characterize hepatitis C virus NS3-4A protease trans-processing activity.
    Author: Hamatake R, Wang HG, Butcher JA, Bifano M, Clark G, Hernandez D, Zhang S, Racela J, Standring D, Colonno R.
    Journal: Intervirology; 1996; 39(4):249-58. PubMed ID: 9078466.
    Abstract:
    An in vitro cleavage system was established to measure HCV NS3 protease trans-processing activity. This system utilizes purified NS3-4A protein from baculovirus, purified substrates expressed by in vitro transcription and translation and defined buffer components. The 41-residue substrates, 5A/5B and 4A/4B, were processed efficiently in trans by wild-type NS3 but not by a catalytically inactive mutant protease; radiolabel sequencing confirmed that NS3-mediated cleavage occurred at the correct cysteine/serine sites, thereby authenticating this system. Two striking features of this in vitro assay are: (1) analogous 4B/5A and 3/4A substrates cannot be processed in trans under the same conditions, and (2) in vitro cleavage of the 5A/5B and 4A/4B sites is highly dependent on the presence of NS4A, which we show is not the case in vivo.
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