Mengo virus 3C proteinase: recombinant expression, intergenus substrate cleavage and localization in vivo.
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.
Hall
D J
DJ
Institute for Molecular Virology, University of Wisconsin-Madison 53706, USA.
Palmenberg
A C
AC
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000914:Antibodies, Viral / Q000378:metabolism; D002213:Capsid / Q000378:metabolism; D002712:Chlorides / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008574:Mengovirus / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins; D017967:Zinc Compounds / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D002712:Chlorides; D004791:Enzyme Inhibitors; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D017967:Zinc Compounds; C016837:zinc chloride; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D007930:Leucine; D005033:Ethylmaleimide; C024974:E 64; D007460:Iodoacetamide