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Mengo virus 3C proteinase: Recombinant expression, intergenus substrate cleavage and localizationin vivo
- Source :
- Virus Genes. 13:99-110
- Publication Year :
- 1996
- Publisher :
- Springer Science and Business Media LLC, 1996.
-
Abstract
- Mengo virus 3C proteinase was cloned and expressed to high levels in a bacterial vector system. The protein was solubilized from inclusion bodies then purified to homogeneity (>95%) by ion exchange chromatography. The recombinant enzyme was proteolytically active in cell-free processing assays with a mengo capsid precursor substrate, L-P1-2A, correctly and proficiently cleaving it into L, 1AB, 1C, 1D and 2A protein products. Further analyses with synthetic peptide substrates encompassing the Mengo or rhinovirus-14 2C/3A cleavage sequences, showed the Mengo 3C could recognize and process specific glutamine-glycine sites within these peptides. The reactivity with the rhinovirus peptide was unexpected, because cross-reactivity between a picornavirus 3C enzyme and a protein substrate from different genus of this family has otherwise never been observed. In reciprocal reactions, a rhinovirus-14 3C preparation was unable to cleave the Mengo-derived synthetic peptide substrate. The recombinant Mengo 3C reactions were also characterized with regard to substrate Km, optimum pH and temperature. The protein was additionally used to raise monoclonal antibodies (mAbs) in mice, which in turn localized natural 3C, 3ABC, 3CD and P3 in immunoblots, immunoprecipitations and indirect immunofluorescence assays of Mengo-infected HeLa cells. The monoclonals showed cross-reactivity with 3C and 3C-containing precursors from encephalomyocarditis virus (EMCV), but did not react with 3C proteins from rhinovirus-14 or poliovirus-1M.
- Subjects :
- Picornavirus
medicine.drug_class
Recombinant Fusion Proteins
viruses
medicine.medical_treatment
Molecular Sequence Data
Peptide
Antibodies, Viral
Monoclonal antibody
Inclusion bodies
Substrate Specificity
law.invention
Iodoacetamide
Mice
Viral Proteins
Capsid
Chlorides
Leucine
law
Virology
Genetics
medicine
Animals
Humans
Amino Acid Sequence
Enzyme Inhibitors
Protein Precursors
Molecular Biology
chemistry.chemical_classification
Mice, Inbred BALB C
Protease
biology
3C Viral Proteases
Mengovirus
Antibodies, Monoclonal
General Medicine
biology.organism_classification
Molecular biology
Cysteine Endopeptidases
Enzyme
Biochemistry
chemistry
Ethylmaleimide
Zinc Compounds
Recombinant DNA
Female
Peptides
HeLa Cells
Subjects
Details
- ISSN :
- 1572994X and 09208569
- Volume :
- 13
- Database :
- OpenAIRE
- Journal :
- Virus Genes
- Accession number :
- edsair.doi.dedup.....1768b47107f18bc8c1041978e75f39b5