1887

Abstract

Summary

The 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephalomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys → Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-70-11-2931
1989-11-01
2022-01-23
Loading full text...

Full text loading...

/deliver/fulltext/jgv/70/11/JV0700112931.html?itemId=/content/journal/jgv/10.1099/0022-1317-70-11-2931&mimeType=html&fmt=ahah

References

  1. Abrahamson M., Ritonja A., Brown M. A., Orubb A., Machleidt W., Barrett A. J. 1987; Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human cystatin C and chicken cystatin. Journal of Biological Chemistry 262:9688–9694
    [Google Scholar]
  2. Abrahamson M., Dalboge H., Olafson I., Carlsen S., Grubb A. 1988; Efficient production of native, biologically active human cystatin C by Escherichia coli. FEBS Letters 236:14–18
    [Google Scholar]
  3. Argos P., Kamer G., Nicklin M. J. H., Wimmer E. 1984; Similarity in gene organization and homology between proteins of the animal picornaviruses and a plant comovirus suggest common ancestry of these virus families. Nucleic Acids Research 12:7251–7267
    [Google Scholar]
  4. Arnold E., Luo M., Vriend B., Rossman M. G., Palmenberg A. C., Parks G. D., Nicklin M. J. H., Wimmer E. 1987; Implications of the picornavirus capsid structure for polyprotein processing. Proceedings of the National Academy of Sciences U.S.A. 8421–25
    [Google Scholar]
  5. Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M., Hanada K. 1982; l-trans-epoxysuccinyl-leucylamide-(4-guanidine)butane (E64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochemical Journal 201:189–198
    [Google Scholar]
  6. Barrett A. J., Rawlings N. D., Davies M. E., Machleidt W., Salvesen G., Turk V. 1986; Cysteine proteinase inhibitors of the cystatin superfamily. In Proteinase Inhibitors515–569 Barrett A. J., Salvesen G. Amsterdam: Elsevier;
    [Google Scholar]
  7. Berger A., Schechter I. 1970; Mapping the active site of papain with the aid of peptide substrate and inhibitors. Philosophical Transactions of the Royal Society B257:249–264
    [Google Scholar]
  8. Callahan P. L., Mizutani S., Colonno R. J. 1985; Molecular cloning and complete sequence determination of RNA genome of human rhinovirus type 14. Proceedings of the National Academy of SciencesU.S.A. 82:732–736
    [Google Scholar]
  9. Carroll A. R., Rowlands D. J., Clarke B. E. 1984; The complete nucleotide sequence of the RNA coding for the primary translation product of foot-and-mouth disease. Nucleic Acids Research 12:2461–2472
    [Google Scholar]
  10. Cann A. J., Stanway G., Hauftmann R., Minor P. D., Schild G. C., Clarke L. D., Mountpord R. C., Almond J. W. 1983; Poliovirus type 3: molecular cloning of the genome and nucleotide sequence of the region encoding the proteinase and polymerase proteins. Nucleic Acids Research 11:1267–1281
    [Google Scholar]
  11. Decock B., Billian A. 1986; Human rhinovirus protein synthesis and polyprotein cleavage in infected HeLa-RH cells. Archives of Virology 90:337–342
    [Google Scholar]
  12. Earle J. A. P., Skuce R. A., Fleming C. S., Hoey E. M., Martin S. J. 1988; The complete nucleotide sequence of a bovine enterovirus. Journal of General Virology 69:253–263
    [Google Scholar]
  13. Elliott S. D., Liu T. Y. 1970; The cysteine proteinases; streptococcal proteinase. Methods in Enzymology 19:252
    [Google Scholar]
  14. Hanecak R., Semler B. L., Anderson C. W., Wimmer E. 1982; Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7C inhibit cleavage at glutamine-glycine pairs. Proceedings of the National Academy of SciencesU.S.A. 79:3973–3977
    [Google Scholar]
  15. Hanecak R., Semler B. L., Arlga H., Anderson C. W., Wimmer E. 1984; Expression of a cloned gene segment of poliovirus in Escherichia coli: evidence for autocatalytic production of the viral proteinase. Cell 37:1063–1073
    [Google Scholar]
  16. Hughes P. J., North C., Jellis C. H., Minor P. D., Stanway G. 1988; The nucleotide sequence of human rhinovirus 1B: molecular relationships within the rhinovirus genus. Journal of General Virology 69:49–58
    [Google Scholar]
  17. Ivanoff L. A., Towatari T., Ray J., Korant B. D., Petteway S. R. 1986; Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli. Proceedings of the National Academy of SciencesU.S.A. 835392–5396
    [Google Scholar]
  18. Jenkins O., Booth J. D., Minor P. D., Almond J. W. 1987; The complete nucleotide sequence of coxsackievirus B4 and its comparison to other members of the picornaviridae. Journal of General Virology 68:1835–1848
    [Google Scholar]
  19. Jupp R. A., Richards A. D., Kay J., Dunn B. M., Wyckoff J. B., Samloff I. M., Yamamoto K. 1988; Identification of the aspartic proteinases from human erythrocyte membranes and gastric mucosa as catalytically equivalent to cathepsin E. Biochemical Journal 254:895–898
    [Google Scholar]
  20. Kamphuis I. G., Drenth J., Baker E. N. 1985; Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. Journal of Molecular Biology 182:317–329
    [Google Scholar]
  21. Kitamura N., Semler B. L., Rothberg P. G., Larsen G. R., Adler C. J., Dorner A. J., Emini E. A., Hanecak R., Lee J. J., Van Der Werf S., Anderson C. W., Wimmer E. 1981; Primary structure, gene organisation and polypeptide expression of poliovirus RNA. Nature London: 291547–553
    [Google Scholar]
  22. Klump W., Marquardt O., Hofschneider P. H. 1984; Biologically active protease of foot-and-mouth disease virus is expressed from cloned cDNA in Escherichia coli . Proceedings of the National Academy of SciencesU.S.A. 81:3351–3353
    [Google Scholar]
  23. Knott J. A., Orr D. C., Montgomery D. S., Sullival C. A., Weston A. 1989; The expression and purification of human rhinovirus protease 3C. European Journal of Biochemistry 182:547–555
    [Google Scholar]
  24. Korant B. D., Towatari T., Ivanoff L., Petteway S., Brzin J., Lenarcic B., Turk V. 1986; Viral therapy - prospects for protease inhibitors. Journal of Cellular Biochemistry 32:91–95
    [Google Scholar]
  25. Korant B. D., Ivanoff L., Towatari T., Cordova A., Kettner C., Turk V., Petteway S. 1986; Control of viral protease action. Journal of Cellular Biochemistry S10A:275
    [Google Scholar]
  26. Larson H. E., Reed S. E., Tyrrell D. A. J. 1980; Isolation of rhinoviruses and coronaviruses from 38 colds in adults. Journal of Medical Virology 51:221–229
    [Google Scholar]
  27. Libby R. T., Cosman D., Cooney M. K., Merriam J. E., March C. J., Hopp T. P. 1988; Human rhinovirus 3C protease: cloning and expression of an active form in Escherichia coli. Biochemistry 27:6262–6268
    [Google Scholar]
  28. Liener I. E., Friedenson B. 1970; The cysteine proteases. Methods in Enzymology 19:261–272
    [Google Scholar]
  29. Lomonossoff G. P., Shanks M. 1983; The nucleotide sequence of cowpea mosaic virus B RNA. EMBO Journal 2:2253–2258
    [Google Scholar]
  30. Murachi T. 1970; The cysteine proteases; bromelain enzymes. Methods in Enzymology 19:273–284
    [Google Scholar]
  31. Nicklin M. J. H., Toyoda H., Murray M. G., Wimmer E. 1986; Proteolytic processing in the replication of polio and related viruses. Bio I Technology 4:33–12
    [Google Scholar]
  32. Nicklin M. J. H., Krai-Asslich H. G., Toyoda H., Dunn J. J., Wimmer E. 1987; Poliovirus polypeptide recursors: expressionin vitro and processing by exogenous 3C and 2A proteinases. Proceedings of the National Academy of SciencesU.S.A. 844002–4006
    [Google Scholar]
  33. Pallansch M. A., Kew O. M., Semler B. L., Omilianowski D. R., Anderson C. W., Wimmler E., Rueckert R. R. 1984; Protein processing map of poliovirus. Journal of Virology 49:873–880
    [Google Scholar]
  34. Palmenberg A. C. 1981; In vitro synthesis and assembly of picomaviralcapsid intermediate structures. Journal of Virology 44:900–906
    [Google Scholar]
  35. Palmenberg A. C. 1987; Picornaviral processing: some new ideas. Journal of Cellular Biochemistry 33:191–198
    [Google Scholar]
  36. Parks G. D., Palmenberg A. C. 1987; Site-specific mutations at a picornavirus VP3/VP1 cleavage site disrupt in vitro processing and assembly of capsid precursors. Journal of Virology 61:3680–3687
    [Google Scholar]
  37. Parks G. D., Duke G. M., Palmenberg A. C. 1986; Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5′non-coding sequences for the P3 region. Journal of Virology 60:376–384
    [Google Scholar]
  38. Place G. A., Chetland J., Galpin I. J., Beynon R. J. 1987; The effects of analogues of chymostatin on lysosomal and non-lysosomal components of protein degradation in isolated hepatocytes. Biochemical and Biophysical Research Communications 925:185–193
    [Google Scholar]
  39. Polgar L., Halasz P. 1982; Current problems in mechanistic studies of serine and cysteine proteinases. Biochemical Journal 207:1–10
    [Google Scholar]
  40. Powers J. C., Harper J. W. 1986; Inhibitors of metalloproteases. In Proteinase Inhibitors219–298 Barrett A. J., Salvesen G. Amsterdam: Elsevier;
    [Google Scholar]
  41. Powers J. C., Harper J. W. 1986; Inhibitors of serine proteinases. In Proteinase Inhibitors55–152 Barrett A. J., Salvesen G. Amsterdam: Elsevier;
    [Google Scholar]
  42. Rich D. H. 1986; Inhibitors of cysteine proteinases. In Proteinase Inhibitors153–178 Barrett A. J., Salvesen G. Amsterdam: Elsevier;
    [Google Scholar]
  43. Robertson B. H., Grubman M. J., Wedell G. N., Moore D. M., Fischer T., Dowbenko D. J., Yansura D. G., Small B., Kleid D. G. 1985; Nucleotide and amino-acid sequence coding for polypeptides of foot-and-mouth disease virus type A12. Journal of Virology 54:651–660
    [Google Scholar]
  44. Rueckert R. R., Wimmer E. 1984; Systematic nomenclature of picornavirus proteins. Journal of Virology 50:957–959
    [Google Scholar]
  45. Semler B. L., Hanecak R., Anderson C. W., Wimmer E. 1981; Cleavage sites in the polypeptide precursors of poliovirus protein P2-X. Virology 114:589–594
    [Google Scholar]
  46. Semler B. L., Johnson V. H., Dewalt P. B., Ypma-Wong M. F. 1987; Site-specific mutagenesis of cDNA clones expressing a poliovirus proteinase. Journal of Cellular Biochemistry 33:39–51
    [Google Scholar]
  47. Shih C-Y. T., Shih D. S. 1981; Cleavage of the capsid protein precursors of encephalomyocarditis virus in rabbit reticulocyte lysates. Journal of Virology 40:942–945
    [Google Scholar]
  48. Shih C-Y. T., Naseer N., Shih D. S. 1982; Rapid method for the preparation of encephalomyocarditis virusm protease from rabbit reticulocyte lysates. Journal of Virology 42:1127–1130
    [Google Scholar]
  49. Shih D. S., Shih C-Y. T., Kew O. M., Pallansch M. A., Rueckert R. R., Kaesberg P. 1978; Cell-free synthesis and processing of the proteins of poliovirus. Proceedings of the National Academy of SciencesU.S.A. 755807–5811
    [Google Scholar]
  50. Shih D. S., Shih C-Y. T., Zimmern D., Rueckert R. R., Kaesberg P. 1979; Translation of encephalomyocarditis virus RNA in reticulocyte lysates: kinetic analysis of the formation of virion proteins and a protein required for processing. Journal of Virology 30:472–480
    [Google Scholar]
  51. Skern T., Sommergruber W., Blaas D., Pieler C., Kuechler E. 1984; Relationship of human rhinovirus strain 2 and poliovirus as indicated by comparison of the polymerase gene regions. Virology 136:125–132
    [Google Scholar]
  52. Skern T., Sommergruber W., Blaas D., Grundler P., Fraundorfer F., Pieler C., Fogy I., Kuechler E. 1985; Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid region. Nucleic Acids Research 13:2111–2126
    [Google Scholar]
  53. Stanway G., Cann A. J., Hauptmann R., Hughes P., Clarke L. D., Mountford R. C., Minor P. D., Schild G. C., Almond J. W. 1983; The nucleotide sequence of poliovirus type 3 Leon 12a1b: comparison with poliovirus type 1. Nucleic Acids Research 11:5629–5643
    [Google Scholar]
  54. Stanway G., Hughes P., Mountford R. C., Minor P. D., Almond J. W. 1984; The complete nucleotide sequence of a common cold virus: human rhinovirus 14. Nucleic Acids Research 12:7859–7875
    [Google Scholar]
  55. Toyoda H., Nicklin M. J. H., Murray M. G., Wimmer E. 1986a; Proteolytic processing of the poliovirus polyprotein by two virus-encoded proteinases. In Protein Engineering Applications in Science, Medicine and Industry319–337 Inouye M., Sarma R. Academic Press; New York:
    [Google Scholar]
  56. Toyoda H., Nicklin M. J. H., Murray M. G., Anderson C. W., Dunn J. J., Studier F. W., Wimmer E. 1986b; A second virus-encoded proteinase involved in the proteolytic processing of poliovirus polyprotein. Cell 45:761–770
    [Google Scholar]
  57. Vos P., Verver J., Van Wezenbeek P., Van Kammen A., Goldbach R. 1984; Study of genetic organisation of a plant viral RNA genome by in vitro expression of a full-length DNA copy. EMBO Journal 3:3049–3053
    [Google Scholar]
  58. Vos P., Jaegle M., Wellink J., Verver J., Eggen R., Van kammen A., Goldbach R. 1987; Infectious RNA transcripts derived from full-length DNA copies of the genomic RNAs of cowpea mosaic virus. Virology 165:33–41
    [Google Scholar]
  59. Wimmer E. 1982; Genome-linked proteins of viruses. Cell 28:199–201
    [Google Scholar]
  60. Yogo Y., Wimmer E. 1972; Polyadenylic acid at the 3′ terminus of poliovirus RNA. Proceedings of the National Academy of SciencesU.S.A. 691877–1882
    [Google Scholar]
  61. Ypma-Wong M. F., Semler B. L. 1987a; Processing determinants required for in vitro cleavage of the poliovirus PI precursor to capsid proteins. Journal of Virology 61:3181–3189
    [Google Scholar]
  62. Ypma-Wong M. F., Semler B. L. 1987b; In vitro molecular genetics as a tool for determining the differential cleavage specificities of the poliovirus 3C proteinase. Nucleic Acids Research 15:2069–2088
    [Google Scholar]
  63. Ypma-Wong M. F., Filman D. J., M Hogle J. M., Semler B. L. 1988; Structural domains of the poliovirus polyprotein are major determinants for proteolytic cleavage at Gln-Gly pairs. Journal of Biological Chemistry 263:17846–17856
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-70-11-2931
Loading
/content/journal/jgv/10.1099/0022-1317-70-11-2931
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error