1887

Abstract

The amino acid sequence of the poliovirus 2C protein contains two highly conserved stretches, GSPGTGKS and MDD, which correspond to the consensus ‘A’ and ‘B’ motifs (GXXXXGKS/T and DD/E, respectively) found in nucleoside triphosphate-binding proteins. To assess the functional importance of these amino acid sequences, we changed conserved and non-conserved amino acids. The replacement of the non-conserved Thr residue with Ser or Ala did not markedly change the virus phenotype. Similarly, replacement of the non-conserved Pro residue by Ala did not abolish virus viability, but changes of this residue to Thr or Asn were not tolerated. No viable mutant could be isolated after transfection of cultured cells with transcripts mutated at the conserved Lys, Ser or Asp residues. However, true revertants were selected from Arg and Ser mutants, from Glu and Gly mutants, and from Ala mutants. Thr mutants not only gave rise to true revertants, but also to two independent isolates of a suppressor mutant, Asn→Tyr. All the lethal mutations resulted in severe inhibition of viral RNA synthesis , although no translational deficiency was detected in a cell-free system. This is the first direct evidence for the functional significance of the nucleoside triphosphatebinding pattern in the poliovirus 2C protein.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-73-8-1977
1992-08-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/jgv/73/8/JV0730081977.html?itemId=/content/journal/jgv/10.1099/0022-1317-73-8-1977&mimeType=html&fmt=ahah

References

  1. Agol V. I., Grachev V. P., Drosdov S. G., Kolesnikova M. S., Kozlov V. G., Ralph N. M., Romanova L. I., Tolskaya E. A., Tyufanov A. V., Viktorova E. G. 1984; Construction and properties of intertypic poliovirus recombinants: first approximation mapping of the major determinants of neurovirulence. Virology 136:41–55
    [Google Scholar]
  2. Agut H., Matsukura T., Belloq C., Dreano M., Nicolas J. G., Girard M. 1981; Isolation and preliminary characterisation of temperature-sensitive mutants of poliovirus type 1. Annales de l’Institut Pasteur/Virologie 132E:445–460
    [Google Scholar]
  3. Argos P., Kamer G., Nicklin M. J. H., Wimmer E. 1984; Similarity in gene organization and homology between proteins of animal picomaviruses and a plant comovirus suggest common ancestry of these virus families. Nucleic Acids Research 12:7251–7267
    [Google Scholar]
  4. Baltera R. F., Tershak D. R. 1989; Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C. Journal of Virology 63:4441–4444
    [Google Scholar]
  5. Bernstein H. D., Sarnow P., Baltimore D. 1986; Genetic complementation among poliovirus mutants derived from an infectious cDNA clone. Journal of Virology 60:1040–1049
    [Google Scholar]
  6. Caliguiri L. A., Tamm I. 1968; Action of guanidine on the replication of poliovirus RNA. Virology 35:408–417
    [Google Scholar]
  7. Caliguiri L. A., Tamm I. 1973; Guanidine and 2-(α-hydroxy-benzyl)benzimidazole (HBB): selective inhibitors of picomavirus multiplication. In Selective Inhibitors of Viral Functions pp. 257–294 Edited by Carter W. Boca Raton: CRC Press;
    [Google Scholar]
  8. Dever T. E., Glynias M. J., Merrick W. C. 1987; GTP-binding domain: three consensus elements with distinct spacing. Proceedings of the National Academy of Sciences, U.S.A. 84:1814–1818
    [Google Scholar]
  9. De Vos A. M., Tong L., Milburn M. B., Matias P. M., Jancarik J., Noguchi S., Nishimura S., Miura K., Ohtsuka E., Kim S.-H. 1988; Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science 239:888–893
    [Google Scholar]
  10. Fichot O., Girard M. 1990; An improved method for sequencing of RNA templates. Nucleic Acids Research 18:6162
    [Google Scholar]
  11. Franssen H., Leunissen J., Goldbach R., Lomonossoff G., Zimmern D. 1984; Homologous sequences in non-structural proteins from cowpea mosaic virus and picomaviruses. EMBO Journal 3:855–861
    [Google Scholar]
  12. Gorbalenya A. E., Koonin E. V. 1989; Vims proteins containing the purine NTP-binding pattern. Nucleic Acids Research 17:8413–8440
    [Google Scholar]
  13. Gorbalenya A. E., Blinov V. M., Koonin E. V. 1985; Prediction of nucleic acid-binding properties of vims proteins from their amino acid sequences. Molekulamaya Genetika 11:30–36
    [Google Scholar]
  14. Gorbalenya A. E., Koonin E. V., Wolf Y. I. 1990; A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA vimses. FEBS Letters 262:145–148
    [Google Scholar]
  15. Im D. S., Muzyczka N. 1990; The AAV origin binding protein Rep 68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61:447–457
    [Google Scholar]
  16. Johnson K. L., Sarnow P. 1991; Three poliovims 2B mutants exhibit noncomplementable defects in viral RNA amplification and display dosage-dependent dominance over wild-type poliovims. Journal of Virology 65:4341–4349
    [Google Scholar]
  17. Jurnak F. 1985; Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science 230:32–36
    [Google Scholar]
  18. Kean K. M., Wychowski C., Kopecka H., Girard M. 1986; Highly infectious plasmids carrying poliovirus cDNA are capable of replication in transfected simian cells. Journal of Virology 59:490–493
    [Google Scholar]
  19. Kean K. M., Agut H., Fichot O., Girard M. 1989; Substitution in the poliovirus replicase gene determines actinomycin D sensitivity of viral replication at elevated temperature. Virus Research 12:19–32
    [Google Scholar]
  20. Kean K. M., Teterina N. L., Marc D., Girard M. 1991; Analysis of putative active site residues of the poliovirus 3C protease. Virology 181:609–619
    [Google Scholar]
  21. Kräusslich H. G., Nicklin M. J. H., Toyoda H., Etchinson D., Wimmer E. 1987; Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220. Journal of Virology 61:2711–2718
    [Google Scholar]
  22. Laín S., Riechmann J. L., García J. A. 1990; RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acids Research 18:7003–7006
    [Google Scholar]
  23. Laín S., Martín M. T., Riechmann J. L., García J. A. 1991; Novel catalytic activity associated with positive-strand RNA virus infection: nucleic acid-stimulated ATPase activity of the plum pox potyvirus helicaselike protein. Journal of Virology 65:1–6
    [Google Scholar]
  24. Levinson A. D. 1986; Normal and activated ras oncogenes and their encoded products. Trends in Genetics 2:81–85
    [Google Scholar]
  25. Li J. P., Baltimore D. 1988; Isolation of poliovirus 2C mutants defective in viral RNA synthesis. Journal of Virology 61:4016–4021
    [Google Scholar]
  26. Marc D., Drugeon G., Haenni A.-L., Girard M., van der Werf S. 1989; Role of myristoylation of poliovirus capsid protein VP4 as determined by site-directed mutagenesis of its N-terminus sequence. EMBO Journal 8:2661–2668
    [Google Scholar]
  27. Neil J. D. 1990; Nucleotide sequence of a region of the feline calicivirus genome which encodes picomavirus-like RNA-dependent RNA polymerase, cysteine protease and 2C polypeptide. Virus Research 17:145–160
    [Google Scholar]
  28. Nobel J., Levintow L. 1970; Dynamics of poliovirus specific RNA synthesis and the effects of inhibitors of virus replication. Virology 42:634–642
    [Google Scholar]
  29. Pallansch M. A., Kew O. M., Semler B. L., Omilianowsky D. R., Anderson C. W., Wimmer E., Rueckert R. R. 1984; Protein processing map of poliovirus. Journal of Virology 49:873–880
    [Google Scholar]
  30. Pincus S., Wimmer E. 1986; Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity. Journal of Virology 60:793–796
    [Google Scholar]
  31. Pincus S., Diamond D., Emini E., Wimmer E. 1986; Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C. Journal of Virology 57:638–646
    [Google Scholar]
  32. Pincus S., Rohl H., Wimmer E. 1987; Guanidine-dependent mutants of poliovirus: identification of three classes with different growth requirements. Virology 157:83–88
    [Google Scholar]
  33. Racaniello V. R., Meriam C. 1986; Poliovirus temperature-sensitive mutant containing a single nucleotide deletion in the 5′-noncoding region of the viral RNA. Virology 155:498–507
    [Google Scholar]
  34. Reuer Q., Kuhn R. J., Wimmer E. 1990; Characterization of poliovirus clones containing lethal and nonlethal mutations in the genome-linked VPg. Journal of Virology 64:2967–2975
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual 2nd edn New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Schaffer F. L., Gordon M. 1966; Differential inhibitory effects of actinomycin D among strains of poliovirus. Journal of Bacteriology 91:2309–2316
    [Google Scholar]
  37. Seeley T. W., Grossman L. 1989; Mutations in the Escherichia coli UvrB ATPase motif compromise excision repair capacity. Proceedings of the National Academy of Sciences, U.S.A. 86:6577–6581
    [Google Scholar]
  38. Stahl H., Droge P., Knippers R. 1986; DNA helicase activity of SV-40 large tumor antigen. EMBO Journal 5:1939–1944
    [Google Scholar]
  39. Tolskaya E. A., Lipskaya G. Yu., Voroshilova M. K., Agol V. I. 1968; Study of temperature-dependent inhibition of reproduction of some picomaviruses by aurantine and mitomycin. Archiv für die gesamte Virusforschung 25:160–176
    [Google Scholar]
  40. 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. Cell 45:761–770
    [Google Scholar]
  41. Valencia A., Chardin P., Wittinghofer A., Sander C. 1991; The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry 30:4637–4648
    [Google Scholar]
  42. van der Werf S., Bradley J., Wimmer E., Studier W. F., Dunn J. J. 1986; Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proceedings of the National Academy of Sciences, U.S.A 83:2330–2334
    [Google Scholar]
  43. Walker J. E., Saraste M., Runswick M. J., Gay N. J. 1982; Distantly related sequences in the α- and β-subunits of ATP-synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO Journal 1945–951
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-73-8-1977
Loading
/content/journal/jgv/10.1099/0022-1317-73-8-1977
Loading

Data & Media loading...

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