Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1987 Oct;61(10):3199–3207. doi: 10.1128/jvi.61.10.3199-3207.1987

Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts.

V N Vakharia, M A Devaney, D M Moore, J J Dunn, M J Grubman
PMCID: PMC255898  PMID: 3041041

Abstract

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.

Full text

PDF
3199

Images in this article

3201Image
on p.3201
3202Image
on p.3202
3203Image
on p.3203
3204Image
on p.3204
3204Image
on p.3204

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnold E., Luo M., Vriend G., Rossmann M. G., Palmenberg A. C., Parks G. D., Nicklin M. J., Wimmer E. Implications of the picornavirus capsid structure for polyprotein processing. Proc Natl Acad Sci U S A. 1987 Jan;84(1):21–25. doi: 10.1073/pnas.84.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beck E., Forss S., Strebel K., Cattaneo R., Feil G. Structure of the FMDV translation initiation site and of the structural proteins. Nucleic Acids Res. 1983 Nov 25;11(22):7873–7885. doi: 10.1093/nar/11.22.7873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black D. N. Proteins induced in BHK cells by infection with foot-and-mouth desease virus. J Gen Virol. 1975 Jan;26(1):109–119. doi: 10.1099/0022-1317-26-1-109. [DOI] [PubMed] [Google Scholar]
  4. Burroughs J. N., Sangar D. V., Clarke B. E., Rowlands D. J., Billiau A., Collen D. Multiple proteases in foot-and-mouth disease virus replication. J Virol. 1984 Jun;50(3):878–883. doi: 10.1128/jvi.50.3.878-883.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Butterworth B. E., Korant B. D. Characterization of the large picornaviral polypeptides produced in the presence of zinc ion. J Virol. 1974 Aug;14(2):282–291. doi: 10.1128/jvi.14.2.282-291.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carroll A. R., Rowlands D. J., Clarke B. E. The complete nucleotide sequence of the RNA coding for the primary translation product of foot and mouth disease virus. Nucleic Acids Res. 1984 Mar 12;12(5):2461–2472. doi: 10.1093/nar/12.5.2461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davanloo P., Rosenberg A. H., Dunn J. J., Studier F. W. Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2035–2039. doi: 10.1073/pnas.81.7.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  9. Forss S., Strebel K., Beck E., Schaller H. Nucleotide sequence and genome organization of foot-and-mouth disease virus. Nucleic Acids Res. 1984 Aug 24;12(16):6587–6601. doi: 10.1093/nar/12.16.6587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goeddel D. V., Heyneker H. L., Hozumi T., Arentzen R., Itakura K., Yansura D. G., Ross M. J., Miozzari G., Crea R., Seeburg P. H. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature. 1979 Oct 18;281(5732):544–548. doi: 10.1038/281544a0. [DOI] [PubMed] [Google Scholar]
  11. Grubman M. J., Bachrach H. L. Isolation of foot-and-mouth disease virus messenger RNA from membrane-bound polyribosomes and characterization of its 5' and 3' termini. Virology. 1979 Oct 30;98(2):466–470. doi: 10.1016/0042-6822(79)90570-1. [DOI] [PubMed] [Google Scholar]
  12. Grubman M. J., Baxt B., Bachrach H. L. Foot-and-mouth disease virion RNA: studies on the relation between the length of its 3'-poly(A) segment and infectivity. Virology. 1979 Aug;97(1):22–31. doi: 10.1016/0042-6822(79)90369-6. [DOI] [PubMed] [Google Scholar]
  13. Grubman M. J., Baxt B. Translation of foot-and-mouth disease virion RNA and processing of the primary cleavage products in a rabbit reticulocyte lysate. Virology. 1982 Jan 15;116(1):19–30. doi: 10.1016/0042-6822(82)90399-3. [DOI] [PubMed] [Google Scholar]
  14. Grubman M. J., Robertson B. H., Morgan D. O., Moore D. M., Dowbenko D. Biochemical map of polypeptides specified by foot-and-mouth disease virus. J Virol. 1984 May;50(2):579–586. doi: 10.1128/jvi.50.2.579-586.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gubler U., Hoffman B. J. A simple and very efficient method for generating cDNA libraries. Gene. 1983 Nov;25(2-3):263–269. doi: 10.1016/0378-1119(83)90230-5. [DOI] [PubMed] [Google Scholar]
  17. Guo L. H., Wu R. Exonuclease III: use for DNA sequence analysis and in specific deletions of nucleotides. Methods Enzymol. 1983;100:60–96. doi: 10.1016/0076-6879(83)00046-4. [DOI] [PubMed] [Google Scholar]
  18. Hanecak R., Semler B. L., Anderson C. W., Wimmer E. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs. Proc Natl Acad Sci U S A. 1982 Jul;79(13):3973–3977. doi: 10.1073/pnas.79.13.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hanecak R., Semler B. L., Ariga H., Anderson C. W., Wimmer E. Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase. Cell. 1984 Jul;37(3):1063–1073. doi: 10.1016/0092-8674(84)90441-0. [DOI] [PubMed] [Google Scholar]
  20. Hardy M. M., Moore D. M. Neutralization of foot-and-mouth disease virus. I. Sensitization of the 140S virion by antibody also reactive with the 12S protein subunit. J Gen Virol. 1981 Aug;55(Pt 2):415–427. doi: 10.1099/0022-1317-55-2-415. [DOI] [PubMed] [Google Scholar]
  21. Harris T. J., Brown F., Sangar D. V. Differential precipitation of foot and mouth disease virus proteins made in vivo and in vitro by hyperimmune and virus particle guinea pig antisera. Virology. 1981 Jul 15;112(1):91–98. doi: 10.1016/0042-6822(81)90615-2. [DOI] [PubMed] [Google Scholar]
  22. Holland J. J., Kiehn E. D. Specific cleavage of viral proteins as steps in the synthesis and maturation of enteroviruses. Proc Natl Acad Sci U S A. 1968 Jul;60(3):1015–1022. doi: 10.1073/pnas.60.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ivanoff L. A., Towatari T., Ray J., Korant B. D., Petteway S. R., Jr Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5392–5396. doi: 10.1073/pnas.83.15.5392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jackson R. J. A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products. Virology. 1986 Feb;149(1):114–127. doi: 10.1016/0042-6822(86)90092-9. [DOI] [PubMed] [Google Scholar]
  25. Jacobson M. F., Baltimore D. Morphogenesis of poliovirus. I. Association of the viral RNA with coat protein. J Mol Biol. 1968 Apr 28;33(2):369–378. doi: 10.1016/0022-2836(68)90195-2. [DOI] [PubMed] [Google Scholar]
  26. Jacobson M. F., Baltimore D. Polypeptide cleavages in the formation of poliovirus proteins. Proc Natl Acad Sci U S A. 1968 Sep;61(1):77–84. doi: 10.1073/pnas.61.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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. Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature. 1981 Jun 18;291(5816):547–553. doi: 10.1038/291547a0. [DOI] [PubMed] [Google Scholar]
  28. Kleid D. G., Yansura D., Small B., Dowbenko D., Moore D. M., Grubman M. J., McKercher P. D., Morgan D. O., Robertson B. H., Bachrach H. L. Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science. 1981 Dec 4;214(4525):1125–1129. doi: 10.1126/science.6272395. [DOI] [PubMed] [Google Scholar]
  29. Klump W., Marquardt O., Hofschneider P. H. Biologically active protease of foot and mouth disease virus is expressed from cloned viral cDNA in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3351–3355. doi: 10.1073/pnas.81.11.3351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Korant B. D. Cleavage of poliovirus-specific polypeptide aggregates. J Virol. 1973 Sep;12(3):556–563. doi: 10.1128/jvi.12.3.556-563.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Liu F. T., Zinnecker M., Hamaoka T., Katz D. H. New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry. 1979 Feb 20;18(4):690–693. doi: 10.1021/bi00571a022. [DOI] [PubMed] [Google Scholar]
  33. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nicklin M. J., Kräusslich H. G., Toyoda H., Dunn J. J., Wimmer E. Poliovirus polypeptide precursors: expression in vitro and processing by exogenous 3C and 2A proteinases. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4002–4006. doi: 10.1073/pnas.84.12.4002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pallansch M. A., Kew O. M., Semler B. L., Omilianowski D. R., Anderson C. W., Wimmer E., Rueckert R. R. Protein processing map of poliovirus. J Virol. 1984 Mar;49(3):873–880. doi: 10.1128/jvi.49.3.873-880.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Palmenberg A. C., Kirby E. M., Janda M. R., Drake N. L., Duke G. M., Potratz K. F., Collett M. S. The nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region. Nucleic Acids Res. 1984 Mar 26;12(6):2969–2985. doi: 10.1093/nar/12.6.2969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Palmenberg A. C., Pallansch M. A., Rueckert R. R. Protease required for processing picornaviral coat protein resides in the viral replicase gene. J Virol. 1979 Dec;32(3):770–778. doi: 10.1128/jvi.32.3.770-778.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Parks G. D., Duke G. M., Palmenberg A. C. Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region. J Virol. 1986 Nov;60(2):376–384. doi: 10.1128/jvi.60.2.376-384.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pelham H. R. Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme. Eur J Biochem. 1978 Apr 17;85(2):457–462. doi: 10.1111/j.1432-1033.1978.tb12260.x. [DOI] [PubMed] [Google Scholar]
  40. Polatnick J., Arlinghaus R. B., Graves J. H., Cowan K. M. Inhibition of cell-free foot-and-mouth disease virus-ribonucleic acid synthesis by antibody. Virology. 1967 Apr;31(4):609–615. doi: 10.1016/0042-6822(67)90189-4. [DOI] [PubMed] [Google Scholar]
  41. Robertson B. H., Grubman M. J., Weddell G. N., Moore D. M., Welsh J. D., Fischer T., Dowbenko D. J., Yansura D. G., Small B., Kleid D. G. Nucleotide and amino acid sequence coding for polypeptides of foot-and-mouth disease virus type A12. J Virol. 1985 Jun;54(3):651–660. doi: 10.1128/jvi.54.3.651-660.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rossmann M. G., Arnold E., Erickson J. W., Frankenberger E. A., Griffith J. P., Hecht H. J., Johnson J. E., Kamer G., Luo M., Mosser A. G. Structure of a human common cold virus and functional relationship to other picornaviruses. Nature. 1985 Sep 12;317(6033):145–153. doi: 10.1038/317145a0. [DOI] [PubMed] [Google Scholar]
  43. Rueckert R. R., Wimmer E. Systematic nomenclature of picornavirus proteins. J Virol. 1984 Jun;50(3):957–959. doi: 10.1128/jvi.50.3.957-959.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sangar D. V., Black D. N., Rowlands D. J., Brown F. Biochemical mapping of the foot-and-mouth disease virus genome. J Gen Virol. 1977 May;35(2):281–297. doi: 10.1099/0022-1317-35-2-281. [DOI] [PubMed] [Google Scholar]
  45. Strebel K., Beck E. A second protease of foot-and-mouth disease virus. J Virol. 1986 Jun;58(3):893–899. doi: 10.1128/jvi.58.3.893-899.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Summers D. F., Maizel J. V., Jr Evidence for large precursor proteins in poliovirus synthesis. Proc Natl Acad Sci U S A. 1968 Mar;59(3):966–971. doi: 10.1073/pnas.59.3.966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Toyoda H., Nicklin M. J., Murray M. G., Anderson C. W., Dunn J. J., Studier F. W., Wimmer E. A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell. 1986 Jun 6;45(5):761–770. doi: 10.1016/0092-8674(86)90790-7. [DOI] [PubMed] [Google Scholar]
  48. Wickens M. P., Buell G. N., Schimke R. T. Synthesis of double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. Optimization for full length second strand synthesis by Escherichia coli DNA polymerase I. J Biol Chem. 1978 Apr 10;253(7):2483–2495. [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES