Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in Escherichia coli.
To determine the P3 region protein-processing sites cleaved by the hepatitis A virus 3C protease, a nested set of constructs containing a portion of 3A (3A* [the asterisk denotes an incomplete protein]), 3B and 3C and various amounts of 3D, fused in frame to Escherichia coli TrpE-coding sequences under control of the tryptophan promoter, was made. Additional plasmids that encoded a portion of 2C (2C*) and the P3 proteins, including complete or incomplete 3D sequences, were constructed. After induction, E. coli containing these recombinant plasmids produced high levels of fusion proteins as insoluble aggregates. 3C-mediated cleavage products were identified by comparison of expression with a matching set of plasmids, containing an engineered mutation in 3C. Cleavage products were detected by immunoblot analyses by using antisera against the TrpE protein, against 3D*, and against 3CD*. Scissile bonds were determined by N-terminal amino acid sequencing of the proteins formed by cleavage. The results showed that when a portion of 2C was present, the primary cleavage by the 3C protease was between 2C and 3A, and the cleavage site was QG, as predicted by J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987. Very little further cleavage of the released P3 protein was detected. When the fusion protein contained no 2C and included only 3A*-to-3D sequences, efficient cleavage occurred between 3B and 3C, at the QS pair, also as predicted by Cohen et al. (J. Virol. 61:50-59, 1987). The latter proteins were also cleaved between 3C and 3D, but less efficiently than between 3B and 3C. Extracts of bacteria expressing proteins from 3A* to 3D also cleaved a radiolabelled hepatitis A virus substrate containing VP1*2ABC* sequences in trans.
Harmon
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SA
Department of Cellular, Viral, and Molecular Biology, University of Utah, School of Medicine, Salt Lake City 84132.
Updike
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W
Jia
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XY
Summers
D F
DF
Ehrenfeld
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E
D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006507:Hepatovirus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins
D011506:Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases