1887

Abstract

Poliovirus proteinase 2A is an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has been obtained that 2A is also involved in genome replication. All enteroviruses have a negatively charged cluster of amino acids at their C terminus (E/ /AMEQ–NH), a common motif suggesting function. When aligned with enterovirus sequences, the 2A proteinase of human rhinovirus type 2 (HRV2) has a shorter C terminus (–NH) and, indeed, the HRV2 2A cannot substitute for poliovirus 2A to yield a viable chimeric virus. Here evidence is provided that the C-terminal cluster of amino acids plays an unknown role in poliovirus genome replication. Deletion of the EEAME sequence from poliovirus 2A is lethal without significantly influencing proteinase function. On the other hand, addition of EAME to HRV2 2A, yielding a C terminus of this enzyme of EAME, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. Poliovirus type 1 Mahoney 2A could be provided to rescue the lethal deletion EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C terminus of poliovirus 2A is involved in particle formation. It is concluded that the C terminus of poliovirus 2A is an essential domain for viral RNA replication but is not essential for proteolytic processing.

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2001-02-01
2020-04-05
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References

  1. Agol V. I., Paul A. V., Wimmer E.. 1999; Paradoxes of the replication of picornaviral genomes. Virus Research62:129–147
    [Google Scholar]
  2. Aldabe R., Feduchi E., Novoa I., Carrasco L.. 1995; Efficient cleavage of p220 by poliovirus 2Apro expression in mammalian cells: effects on vaccinia virus. Biochemical and Biophysical Research Communications215:928–936
    [Google Scholar]
  3. Alexander L., Lu H. H., Wimmer E.. 1994; Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene. Proceedings of the National Academy of Sciences, USA91:1406–1410
    [Google Scholar]
  4. Ansardi D. C., Porter D. C., Morrow C. D.. 1991; Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures. Journal of Virology65:2088–2092
    [Google Scholar]
  5. Ansardi D. C., Porter D. C., Morrow C. D.. 1993; Complementation of a poliovirus defective genome by a recombinant vaccinia virus which provides poliovirus P1 capsid precursor in trans . Journal of Virology67:3684–3690
    [Google Scholar]
  6. Ansardi D. C., Pal-Ghosh R., Porter D., Morrow C. D.. 1995; Encapsidation and serial passage of a poliovirus replicon which expresses an inactive 2A proteinase. Journal of Virology69:1359–1366
    [Google Scholar]
  7. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K.. (editors) 1994; Current Protocols in Molecular Biology New York: John Wiley;
    [Google Scholar]
  8. Badorff C., Lee G. H., Lamphear B. J., Martone M. E., Campbell K. P., Rhoads R. E., Knowlton K. U.. 1999; Enteroviral protease 2A cleaves dystrophin: evidence of cytoskeletal disruption in an acquired cardiomyopathy. Nature Medicine5:320–326
    [Google Scholar]
  9. Barclay W., Li Q., Hutchinson G., Moon D., Richardson A., Percy N., Almond J. W., Evans D. J.. 1998; Encapsidation studies of poliovirus subgenomic replicons. Journal of General Virology79:1725–1734
    [Google Scholar]
  10. Barco A., Carrasco L.. 1995; Poliovirus 2Apro expression inhibits growth of yeast cells. FEBS Letters371:4–8
    [Google Scholar]
  11. Barco A., Feduchi E., Carrasco L.. 2000; A stable HeLa cell line that inducibly expresses poliovirus 2Apro: effects on cellular and viral gene expression. Journal of Virology74:2383–2392
    [Google Scholar]
  12. Bazan J. F., Fletterick R. J.. 1988; Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications. Proceedings of the National Academy of Sciences, USA85:7872–7876
    [Google Scholar]
  13. Bernstein H. D., Sarnow P., Baltimore D.. 1986; Genetic complementation among poliovirus mutants derived from an infectious cDNA clone. Journal of Virology60:1040–1049
    [Google Scholar]
  14. Bovee M. L., Marissen W. E., Zamora M., Lloyd R. E.. 1998a; The predominant elF4G-specific cleavage activity in poliovirus-infected HeLa cells is distinct from 2A protease. Virology245:229–240
    [Google Scholar]
  15. Bovee M. L., Lamphear B. J., Rhoads R. E., Lloyd R. E.. 1998b; Direct cleavage of elF4G by poliovirus 2A protease is inefficient in vitro . Virology245:241–249
    [Google Scholar]
  16. Clever J., Sassetti C., Parslow T. G.. 1995; RNA secondary structure and binding sites for gag gene products in the 5′ packaging signal of human immunodeficiency virus type 1. Journal of Virology69:2101–2109
    [Google Scholar]
  17. Elroy-Stein O., Moss B.. 1990; Cytoplasmic expression system based on constitutive synthesis of bacteriophage T7 RNA polymerase in mammalian cells. Proceedings of the National Academy of Sciences, USA87:6743–6747
    [Google Scholar]
  18. Goldstaub D., Gradi A., Bercovitch Z., Grosmann Z., Nophar Y., Luria S., Sonenberg N., Kahana C.. 2000; Poliovirus 2A protease induces apoptotic cell death. Molecular and Cellular Biology20:1271–1277
    [Google Scholar]
  19. Gromeier M., Alexander L., Wimmer E.. 1996; Internal ribosomal entry site substitution eliminates neurovirulence in intergeneric poliovirus recombinants. Proceedings of the National Academy of Sciences, USA93:2370–2375
    [Google Scholar]
  20. Gromeier M., Wimmer E., Gorbalenya A. E.. 1999; Genetics, pathogenesis, and evolution of picornaviruses. In Origin and Evolution of Viruses pp287–343 Edited by Domingo E., Webster R. G., Holland J. J.. New York: Academic Press;
    [Google Scholar]
  21. Haghighat A., Svitkin Y., Novoa I., Kuechler E., Skern T., Sonenberg N.. 1996; The eIF4G-eIF4E complex is the target for direct cleavage by the rhinovirus 2A proteinase. Journal of Virology70:8444–8450
    [Google Scholar]
  22. Hambidge S. J., Sarnow P.. 1992; Translational enhancement of the poliovirus 5′ noncoding region mediated by virus-encoded polypeptide 2A. Proceedings of the National Academy of Sciences, USA89:10272–10276
    [Google Scholar]
  23. Harmon S. A., Emerson S. U., Huang Y. K., Summers D. F., Ehrenfeld E.. 1995; Hepatitis A viruses with deletions in the 2A gene are infectious in cultured cells and marmosets. Journal of Virology69:5576–5581
    [Google Scholar]
  24. Hellen C. U. T., Fäcke M., Kräusslich H.-G., Lee C.-K., Wimmer E.. 1991; Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220. Journal of Virology65:4226–4231
    [Google Scholar]
  25. Hellen C. U., Lee C. K., Wimmer E.. 1992; Determinants of substrate recognition by poliovirus 2A proteinase. Journal of Virology66:3330–3338
    [Google Scholar]
  26. Joachims M., Van Breugel P. C., Lloyd R. E.. 1999; Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro . Journal of Virology73:718–727
    [Google Scholar]
  27. Kerekatte V., Keiper B. D., Badorff C., Cai A., Knowlton K. U., Rhoads R. E.. 1999; Cleavage of poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo : another mechanism for host protein synthesis shutoff?. Journal of Virology73:709–717
    [Google Scholar]
  28. Kitamura N., Adler C. J., Rothberg P. G., Martinko J., Nathenson S. G., Wimmer E.. 1980; The genome-linked protein of picornaviruses. Vll. Genetic mapping of poliovirus VPg by protein and RNA sequence studies. Cell21:295–302
    [Google Scholar]
  29. 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 organization and polypeptide expression of poliovirus RNA. Nature291:547–553
    [Google Scholar]
  30. Klump H., Auer H., Liebig H. D., Kuechler E., Skern T.. 1996; Proteolytically active 2A proteinase of human rhinovirus 2 is toxic for Saccharomyces cerevisiae but does not cleave the homologues of eIF-4 gamma in vivo or in vitro . Virology220:109–118
    [Google Scholar]
  31. Kräusslich H. G., Nicklin M. J. H., Toyoda H., Etchison D., Wimmer E.. 1987; Poliovirus proteinase 2A induces cleavage of eukaryotic initiation factor 4F polypeptide p220. Journal of Virology61:2711–2718
    [Google Scholar]
  32. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685
    [Google Scholar]
  33. Lamphear B. J., Yan R., Yang F., Waters D., Liebig H. D., Klump H., Kuechler E., Skern T., Rhoads R. E.. 1993; Mapping the cleavage site in protein synthesis initiation factor eIF-4 gamma of the 2A proteases from human coxsackievirus and rhinovirus. Journal of Biological Chemistry268:19200–19203
    [Google Scholar]
  34. Lawson M. A., Semler B. L.. 1990; Picornavirus protein processing – enzymes, substrates, and genetic regulation. Current Topics in Microbiology and Immunology161:49–87
    [Google Scholar]
  35. Lee C.-K., Wimmer E.. 1988; Proteolytic processing of poliovirus polyprotein: elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis. Virology166:405–414
    [Google Scholar]
  36. Lee Y. F., Nomoto A., Detjen B. M., Wimmer E.. 1977; A protein covalently linked to poliovirus genome RNA. Proceedings of the National Academy of Sciences, USA74:59–63
    [Google Scholar]
  37. Liebig H.-D., Ziegler E., Yan R., Hartmuth K., Klump H., Kowalski H., Blaas D., Sommergruber W., Frasel L., Lamphear B., Rhoads R., Kuechler E., Skern T.. 1993; Purification of two picornaviral 2A proteinases: interaction with eIF-4γ and influence on in vitro translation. Biochemistry32:7581–7588
    [Google Scholar]
  38. Lu H. H., Wimmer E.. 1996; Poliovirus chimeras replicating under the translational control of genetic elements of hepatitis C virus reveal unusual properties of the internal ribosomal entry site of hepatitis C virus. Proceedings of the National Academy of Sciences, USA93:1412–1417
    [Google Scholar]
  39. Lu H. H., Alexander L., Wimmer E.. 1995a; Construction and genetic analysis of dicistronic polioviruses containing open reading frames for epitopes of human immunodeficiency virus type 1 gp120. Journal of Virology69:4797–4806
    [Google Scholar]
  40. Lu H. H., Li X., Cuconati A., Wimmer E.. 1995b; Analysis of picornavirus 2Apro proteins: separation of proteinase from translation and replication functions. Journal of Virology69:7445–7452
    [Google Scholar]
  41. Macadam A. J., Stone D. M., Almond J. W., Minor P. D.. 1994; The 5′ noncoding region and virulence of poliovirus vaccine strains. Trends in Microbiology2:449–454
    [Google Scholar]
  42. Mansky L. M., Krueger A. E., Temin H. M.. 1995; The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5′ end of the gag gene. Journal of Virology69:3282–3289
    [Google Scholar]
  43. Martin-Belmonte F., Lopez-Guerrero J. A., Carrasco L., Alonso M. A.. 2000; The amino-terminal nine amino acid sequence of poliovirus capsid VP4 protein is sufficient to confer N -myristoylation and targeting to detergent-insoluble membranes. Biochemistry39:1083–1090
    [Google Scholar]
  44. Michiels T., Dejong V., Rodrigus R., Shaw-Jackson C.. 1997; Protein 2A is not required for Theiler’s virus replication. Journal of Virology71:9549–9556
    [Google Scholar]
  45. Molla A., Paul A. V., Wimmer E.. 1991; Cell-free, de novo synthesis of poliovirus. Science254:1647–1651
    [Google Scholar]
  46. Molla A., Jang S. K., Paul A. V., Reuer Q., Wimmer E.. 1992; Cardioviral internal ribosomal entry site is functional in a genetically engineered dicistronic poliovirus. Nature356:255–257
    [Google Scholar]
  47. Molla A., Hellen C. U. T., Wimmer E.. 1993a; Inhibition of proteolytic activity of poliovirus and rhinovirus 2A proteinases by elastase-specific inhibitors. Journal of Virology67:4688–4695
    [Google Scholar]
  48. Molla A., Paul A. V., Schmid M., Jang S. K., Wimmer E.. 1993b; Studies on dicistronic polioviruses implicate viral proteinase 2Apro in RNA replication. Virology196:739–747
    [Google Scholar]
  49. Mueller S., Wimmer E.. 1998; Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardiovirus-like open reading frames. Journal of Virology72:20–31
    [Google Scholar]
  50. Mueller S., Wimmer E.. 2000; Picornaviruses as tools of antigen delivery. In Viral Vectors: Basic Science and Gene Therapy . pp543–562 Edited by Cid-Arregui A., Garcia-Carranca A.. Natick, MA: Eaton Publishing;
  51. Newman J. F., Piatti P. G., Ryan M. D., Brown F.. 1994; Function of minor polypeptides in foot-and-mouth disease virus and poliovirus. Trends in Microbiology2:494–497
    [Google Scholar]
  52. Nugent C. I., Johnson K. L., Sarnow P., Kirkegaard K.. 1999; Functional coupling between replication and packaging of poliovirus replicon RNA. Journal of Virology73:427–435
    [Google Scholar]
  53. O’Neill R. E., Racaniello V. R.. 1989; Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2. Journal of Virology63:5069–5075
    [Google Scholar]
  54. Palmenberg A. C.. 1989; Sequence alignments of picornavirus capsid proteins. In Molecular Aspects of Picornavirus Infection and Detection pp211–241 Edited by Semler B., Ehrenfeld E.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  55. Petersen J. F., Cherney M. M., Liebig H. D., Skern T., Kuechler E., James M. N.. 1999; The structure of the 2A proteinase from a common cold virus: a proteinase responsible for the shut-off of host-cell protein synthesis. EMBO Journal18:5463–5475
    [Google Scholar]
  56. Pfister T., Mirazayan C., Wimmer E.. 1999; Polioviruses: molecular biology. In Encyclopedia of Virology pp1330–1348 Edited by Granoff A., Webster R.. London: Academic Press;
    [Google Scholar]
  57. Porter D. C., Ansardi D. C., Wang J., McPherson S., Moldoveanu Z., Morrow C. D.. 1998; Demonstration of the specificity of poliovirus encapsidation using a novel replicon which encodes enzymatically active firefly luciferase. Virology243:1–11
    [Google Scholar]
  58. Pringle C. R.. 1999; Virus taxonomy at the XIth International Congress of Virology. Sydney, Australia: 1999; Archives of Virology144:2065–2070
    [Google Scholar]
  59. Reuer Q., Kuhn R. J., Wimmer E.. 1990; Characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked protein VPg. Journal of Virology64:2967–2975
    [Google Scholar]
  60. Rowe A., Ferguson G. L., Minor P. D., Macadam A. J.. 2000; Coding changes in the poliovirus protease 2A compensate for 5′NCR domain V disruptions in a cell-specific manner. Virology269:284–293
    [Google Scholar]
  61. Rueckert R. R.. 1996; Picornaviridae and their replication. In Fundamental Virology pp507–548 Edited by Fields B. N., Knipe D. M., Howley P. M. Philadelphia: Lippincott–Raven;
    [Google Scholar]
  62. Sambrook J., Fritsch E. F., Maniatis T.. 1989; Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  63. Seipelt J., Guarne A., Bergmann E., James M., Sommergruber W., Fita I., Skern T.. 1999; The structure of picornaviral proteinases. Virus Research62:159–168
    [Google Scholar]
  64. Skern T., Sommergruber W., Blaas D., Gruendler P., Fraundorfer F., Pieler C., Fogy I., Kuechler E.. 1985; Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region. Nucleic Acids Research13:2111–2126
    [Google Scholar]
  65. Skern T., Sommergruber W., Auer H., Volkmann P., Zorn M., Liebig H. D., Fessl F., Blaas D., Kuechler E.. 1991; Substrate requirements of a human rhinoviral 2A proteinase. Virology181:46–54
    [Google Scholar]
  66. Sommergruber W., Zorn M., Blaas D., Fessl F., Volkmann P., Maurer-Fogy I., Pallai P., Merluzzi V., Matteo M., Skern T.. and others 1989; Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis. Virology169:68–77
    [Google Scholar]
  67. Sommergruber W., Casari G., Fessl F., Seipelt J., Skern T.. 1994; The 2A proteinase of human rhinovirus is a zinc containing enzyme. Virology204:815–818
    [Google Scholar]
  68. Toyoda H., Nicklin M. J. H., Murray M. G., Anderson C. W., Dunn J. J., Studier F. W., Wimmer E.. 1986; A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. Cell45:761–770
    [Google Scholar]
  69. van der Werf S., Bradley J., Wimmer E., Studier F. W., Dunn J. J.. 1986; Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA83:2330–2334
    [Google Scholar]
  70. Ventoso I., Carrasco L.. 1995; A poliovirus 2Apro mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects. Journal of Virology69:6280–6288
    [Google Scholar]
  71. Ventoso I., Barco A., Carrasco L.. 1998; Mutational analysis of poliovirus 2Apro. Distinct inhibitory functions of 2Apro on translation and transcription. Journal of Biological Chemistry273:27960–27967
    [Google Scholar]
  72. Ventoso I., Barco A., Carrasco L.. 1999; Genetic selection of poliovirus 2Apro-binding peptides. Journal of Virology73:814–818
    [Google Scholar]
  73. Weiss B., Nitschko H., Ghattas I., Wright R., Schlesinger S.. 1989; Evidence for specificity in the encapsidation of Sindbis virus RNAs. Journal of Virology63:5310–5318
    [Google Scholar]
  74. Wessely R., Klingel K., Santana L. F., Dalton N., Hongo M., Lederer W. J., Kandolf R., Knowlton K. U.. 1998; Transgenic expression of replication-restricted enteroviral genomes in heart muscle induces defective excitation-contraction coupling and dilated cardiomyopathy. Journal of Clinical Investigation102:1444–1453
    [Google Scholar]
  75. Wimmer E., Hellen C. U., Cao X.. 1993; Genetics of poliovirus. Annual Review of Genetics27:353–436
    [Google Scholar]
  76. Yalamanchili P., Banerjee R., Dasgupta A.. 1997; Poliovirus-encoded protease 2APro cleaves the TATA-binding protein but does not inhibit host cell RNA polymerase II transcription in vitro . Journal of Virology71:6881–6886
    [Google Scholar]
  77. Yu S. F., Lloyd R. E.. 1991; Identification of essential amino acid residues in the functional activity of poliovirus 2A protease. Virology182:615–625
    [Google Scholar]
  78. Yu S. F., Benton P., Bovee M., Sessions J., Lloyd R. E.. 1995; Defective RNA replication by poliovirus mutants deficient in 2A protease cleavage activity. Journal of Virology69:247–252
    [Google Scholar]
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