Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles
| Autor: | P J McCann, Gregory Yamanaka, S P Weinheimer, Donald R. O'Boyle, M G Cordingley, C.L. DiIanni, Ingrid C. Deckman, B A Boyd, J T Stevens, D A Drier |
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| Rok vydání: | 1993 |
| Předmět: |
Genes
Viral Recombinant Fusion Proteins medicine.medical_treatment Blotting Western Restriction Mapping Immunology Biology Cleavage (embryo) Polymerase Chain Reaction Microbiology Chromatography Affinity Viral Proteins Capsid Thrombin Virology Endopeptidases Centrifugation Density Gradient Escherichia coli Serine medicine Point Mutation Simplexvirus Amino Acid Sequence Cloning Molecular Binding site Peptide sequence Alanine Binding Sites Protease Molecular biology Fusion protein NS2-3 protease Biochemistry Insect Science Mutagenesis Site-Directed Research Article Plasmids medicine.drug |
| Zdroj: | Journal of Virology. 67:5813-5822 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.67.10.5813-5822.1993 |
| Popis: | The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly. |
| Databáze: | OpenAIRE |
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