Establishment of an in vitro assay to characterize hepatitis C virus NS3-4A protease trans-processing activity.
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.
Hamatake
R
R
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492, USA.
Wang
H G
HG
Butcher
J A
JA
Bifano
M
M
Clark
G
G
Hernandez
D
D
Zhang
S
S
Racela
J
J
Standring
D
D
Colonno
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D016174:Hepacivirus / Q000201:enzymology*; D006801:Humans; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases