Purification and partial characterization of poliovirus protease 2A by means of a functional assay.
H König,B Rosenwirth +1 more
TLDR
Inhibition studies with known protease inhibitors revealed that poliovirus protease 2A is probably a sulfhydryl protease, which was optimal near neutral pH and had an extremely short half-life at physiological temperatures.Abstract:
The purification of poliovirus protease 2A from infected cells by a functional assay is described. A small synthetic peptide was cleaved specifically by an esterase present in poliovirus-infected cells. Since the enzyme proved extremely unstable in crude extracts a rapid and efficient purification procedure had to be developed. By treatment with different detergents followed by high-speed centrifugation, the esterase activity was separated from inactivating cellular enzymes and was solubilized. Purification to more than 90% homogeneity could be achieved by a single chromatography step, namely, by gel filtration through Superose 12 under fast-protein liquid chromatography conditions. The esterase activity was associated with a protein of 17,000 daltons and copurified with poliovirus protein 2A. Furthermore, antibodies to 2A specifically precipitated the esterase activity. Thus, the esterase was identified as poliovirus protease 2A. Inhibition studies with known protease inhibitors revealed that 2A is probably a sulfhydryl protease. Of the metal ions tested, only zinc exerted significant inhibitory effects. The esterase activity was optimal near neutral pH and had an extremely short half-life at physiological temperatures.read more
Citations
More filters
Journal ArticleDOI
Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.
W G Dougherty,Bert L. Semler +1 more
TL;DR: The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors.
Journal ArticleDOI
Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus and rhinovirus.
Barry J. Lamphear,Riqiang Yan,Fang Yang,Debra A. Waters,Hans Dieter Liebig,Hannes Klump,Ernst Kuechler,Tim Skern,Robert E. Rhoads +8 more
TL;DR: This report has used highly purified recombinant 2A protease from either human Coxsackievirus serotype B4 or rhinovirus serotypes 2 to cleave eIF-4 gamma in vitro in the eif-4 complex purified from rabbit reticulocytes, and found neither the rate of cleavage nor fragment sizes were affected by addition of e IF-3.
Journal ArticleDOI
Virus-encoded proteinases of the picornavirus super-group.
Martin D. Ryan,Mike Flint +1 more
TL;DR: This paper presents a meta-anatomy of the immune response to central giant coronavirus, a model that has shown promise in understanding the immune system’s response to infectious disease.
Journal ArticleDOI
Selective inhibitors of picornavirus replication.
TL;DR: The current review aims to highlight the most important developments during the past decades in the search for antivirals against picornaviruses.
Journal ArticleDOI
Purification of two picornaviral 2A proteinases: Interaction with eIF-4.gamma. and influence on the in vitro translation
Hans-Dieter Liebig,E Ziegler,Yan R,Hartmuth K,Klump H,Kowalski H,Dieter Blaas,Sommergruber W,Frasel L,Barry J. Lamphear +9 more
TL;DR: A mammalian cell infected with a human rhinovirus or enterovirus has a much reduced capability to translate capped mRNAs (the host cell shutoff), while still allowing translation of uncapped viral RNA, so mature proteinases 2A of human rhInovirus 2 and coxsackievirus B4 were expressed in soluble form in Escherichia coli.
References
More filters
Journal ArticleDOI
Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Journal Article
Cleavage of structural proteins during the assemble of the head of bacterio-phage T4
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Journal ArticleDOI
Amino Acid Metabolism in Mammalian Cell Cultures
TL;DR: The present article "is a progress report rather than a review and in large part summarizes studies from a single laboratory" on the minimal essential medium for cultivation of mammalian cells in either monolayer or suspension.
Journal ArticleDOI
Plaque formation and isolation of pure lines with poliomyelitis viruses.
Renato Dulbecco,Marguerite Vogt +1 more
TL;DR: Pure virus lines were established by isolating the virus population produced in single plaques, which had the same morphological, serological, and pathogenic properties as the parent strain.
Journal ArticleDOI
Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins
TL;DR: A new silver stain for electrophoretically separated polypeptides can be rapidly and easily used and can detect as little as 0.01 nanogram of protein per square millimeter when employed with two-dimensional electrophoresis.
Related Papers (5)
Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.
J F Bazan,Robert J. Fletterick +1 more