1887

Abstract

A stem–loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a -acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication and abolished uridylylation of VPg . Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, RNNNAARNNNNNNR, is also applicable to CVB3 CRE(2C) and that a positive correlation exists between the ability of CRE(2C) mutants to serve as template in the uridylylation reaction and the capacity of these mutants to support viral RNA replication. To further investigate the effects of the mutations on negative-strand RNA synthesis, an translation/replication system containing HeLa S10 cell extracts was used. Similar to the results observed for poliovirus and rhinovirus, it was found that a complete disruption of the CRE(2C) structure interfered with positive-strand RNA synthesis, but not with negative-strand synthesis. All CRE(2C) point mutants affecting the enteroviral CRE consensus loop, however, showed a marked decrease in efficiency to induce negative-strand synthesis. Moreover, a transition (AG) regarding the first templating adenosine residue in the loop was even unable to initiate complementary negative-strand synthesis above detectable levels. Taken together, these results indicate that the CVB3 CRE(2C) is not only required for the initiation of positive-strand RNA synthesis, but also plays an essential role in the efficient initiation of negative-strand RNA synthesis, a conclusion that has not been reached previously by using the cell-free system.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.81297-0
2006-01-01
2019-11-18
Loading full text...

Full text loading...

/deliver/fulltext/jgv/87/1/103.html?itemId=/content/journal/jgv/10.1099/vir.0.81297-0&mimeType=html&fmt=ahah

References

  1. Andino, R., Rieckhof, G. E. & Baltimore, D. ( 1990; ). A functional ribonucleoprotein complex forms around the 5′ end of poliovirus RNA. Cell 63, 369–380.[CrossRef]
    [Google Scholar]
  2. Barton, D. J., Black, E. P. & Flanegan, J. B. ( 1995; ). Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA. J Virol 69, 5516–5527.
    [Google Scholar]
  3. Barton, D. J., O'Donnell, B. J. & Flanegan, J. B. ( 2001; ). 5′ cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. EMBO J 20, 1439–1448.[CrossRef]
    [Google Scholar]
  4. Crowder, S. & Kirkegaard, K. ( 2005; ). Trans-dominant inhibition of RNA viral replication can slow growth of drug-resistant viruses. Nat Genet 37, 701–709.[CrossRef]
    [Google Scholar]
  5. Gamarnik, A. V. & Andino, R. ( 1998; ). Switch from translation to RNA replication in a positive-stranded RNA virus. Genes Dev 12, 2293–2304.[CrossRef]
    [Google Scholar]
  6. Gerber, K., Wimmer, E. & Paul, A. V. ( 2001; ). Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2Apro. J Virol 75, 10979–10990.[CrossRef]
    [Google Scholar]
  7. Gohara, D. W., Ha, C. S., Kumar, S., Ghosh, B., Arnold, J. J., Wisniewski, T. J. & Cameron, C. E. ( 1999; ). Production of “authentic” poliovirus RNA-dependent RNA polymerase (3Dpol) by ubiquitin–protease-mediated cleavage in Escherichia coli. Protein Expr Purif 17, 128–138.[CrossRef]
    [Google Scholar]
  8. Goodfellow, I., Chaudhry, Y., Richardson, A., Meredith, J., Almond, J. W., Barclay, W. & Evans, D. J. ( 2000; ). Identification of a cis-acting replication element within the poliovirus coding region. J Virol 74, 4590–4600.[CrossRef]
    [Google Scholar]
  9. Goodfellow, I. G., Kerrigan, D. & Evans, D. J. ( 2003a; ). Structure and function analysis of the poliovirus cis-acting replication element (CRE). RNA 9, 124–137.[CrossRef]
    [Google Scholar]
  10. Goodfellow, I. G., Polacek, C., Andino, R. & Evans, D. J. ( 2003b; ). The poliovirus 2C cis-acting replication element-mediated uridylylation of VPg is not required for synthesis of negative-sense genomes. J Gen Virol 84, 2359–2363.[CrossRef]
    [Google Scholar]
  11. Herold, J. & Andino, R. ( 2000; ). Poliovirus requires a precise 5′ end for efficient positive-strand RNA synthesis. J Virol 74, 6394–6400.[CrossRef]
    [Google Scholar]
  12. Herold, J. & Andino, R. ( 2001; ). Poliovirus RNA replication requires genome circularization through a protein–protein bridge. Mol Cell 7, 581–591.[CrossRef]
    [Google Scholar]
  13. Klump, W. M., Bergmann, I., Müller, B. C., Ameis, D. & Kandolf, R. ( 1990; ). Complete nucleotide sequence of infectious coxsackievirus B3 cDNA: two initial 5′ uridine residues are regained during plus-strand RNA synthesis. J Virol 64, 1573–1583.
    [Google Scholar]
  14. Lobert, P.-E., Escriou, N., Ruelle, J. & Michiels, T. ( 1999; ). A coding RNA sequence acts as a replication signal in cardioviruses. Proc Natl Acad Sci U S A 96, 11560–11565.[CrossRef]
    [Google Scholar]
  15. Lyons, T., Murray, K. E., Roberts, A. W. & Barton, D. J. ( 2001; ). Poliovirus 5′-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis. J Virol 75, 10696–10708.[CrossRef]
    [Google Scholar]
  16. Mason, P. W., Bezborodova, S. V. & Henry, T. M. ( 2002; ). Identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus. J Virol 76, 9686–9694.[CrossRef]
    [Google Scholar]
  17. McKnight, K. L. & Lemon, S. M. ( 1998; ). The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication. RNA 4, 1569–1584.[CrossRef]
    [Google Scholar]
  18. Melchers, W. J. G., Hoenderop, J. G. J., Bruins Slot, H. J., Pleij, C. W. A., Pilipenko, E. V., Agol, V. I. & Galama, J. M. D. ( 1997; ). Kissing of the two predominant hairpin loops in the coxsackie B virus 3′ untranslated region is the essential structural feature of the origin of replication required for negative-strand RNA synthesis. J Virol 71, 686–696.
    [Google Scholar]
  19. Melchers, W. J. G., Bakkers, J. M. J. E., Bruins Slot, H. J., Galama, J. M. D., Agol, V. I. & Pilipenko, E. V. ( 2000; ). Cross-talk between orientation-dependent recognition determinants of a complex control RNA element, the enterovirus oriR. RNA 6, 976–987.[CrossRef]
    [Google Scholar]
  20. Merkle, I., van Ooij, M. J. M., van Kuppeveld, F. J. M., Glaudemans, D. H. R. F., Galama, J. M. D., Henke, A., Zell, R. & Melchers, W. J. G. ( 2002; ). Biological significance of a human enterovirus B-specific RNA element in the 3′ nontranslated region. J Virol 76, 9900–9909.[CrossRef]
    [Google Scholar]
  21. Mirmomeni, M. H., Hughes, P. J. & Stanway, G. ( 1997; ). An RNA tertiary structure in the 3′ untranslated region of enteroviruses is necessary for efficient replication. J Virol 71, 2363–2370.
    [Google Scholar]
  22. Molla, A., Paul, A. V. & Wimmer, E. ( 1991; ). Cell-free, de novo synthesis of poliovirus. Science 254, 1647–1651.[CrossRef]
    [Google Scholar]
  23. Morasco, B. J., Sharma, N., Parilla, J. & Flanegan, J. B. ( 2003; ). Poliovirus cre(2C)-dependent synthesis of VPgpUpU is required for positive- but not negative-strand RNA synthesis. J Virol 77, 5136–5144.[CrossRef]
    [Google Scholar]
  24. Murray, K. E. & Barton, D. J. ( 2003; ). Poliovirus CRE-dependent VPg uridylylation is required for positive-strand RNA synthesis but not for negative-strand RNA synthesis. J Virol 77, 4739–4750.[CrossRef]
    [Google Scholar]
  25. Nayak, A., Goodfellow, I. G. & Belsham, G. J. ( 2005; ). Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro. J Virol 79, 7698–7706.[CrossRef]
    [Google Scholar]
  26. Nomoto, A., Detjen, B., Pozzatti, R. & Wimmer, E. ( 1977a; ). The location of the polio genome protein in viral RNAs and its implication for RNA synthesis. Nature 268, 208–213.[CrossRef]
    [Google Scholar]
  27. Nomoto, A., Kitamura, N., Golini, F. & Wimmer, E. ( 1977b; ). The 5′-terminal structures of poliovirion RNA and poliovirus mRNA differ only in the genome-linked protein VPg. Proc Natl Acad Sci U S A 74, 5345–5349.[CrossRef]
    [Google Scholar]
  28. Paul, A. V., van Boom, J. H., Filippov, D. & Wimmer, E. ( 1998; ). Protein-primed RNA synthesis by purified poliovirus RNA polymerase. Nature 393, 280–284.[CrossRef]
    [Google Scholar]
  29. Paul, A. V., Rieder, E., Kim, D. W., van Boom, J. H. & Wimmer, E. ( 2000; ). Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J Virol 74, 10359–10370.[CrossRef]
    [Google Scholar]
  30. Paul, A. V., Yin, J., Mugavero, J., Rieder, E., Liu, Y. & Wimmer, E. ( 2003; ). A “slide-back” mechanism for the initiation of protein-primed RNA synthesis by the RNA polymerase of poliovirus. J Biol Chem 278, 43951–43960.[CrossRef]
    [Google Scholar]
  31. Pettersson, R. F., Ambros, V. & Baltimore, D. ( 1978; ). Identification of a protein linked to nascent poliovirus RNA and to the polyuridylic acid of negative-strand RNA. J Virol 27, 357–365.
    [Google Scholar]
  32. Pilipenko, E. V., Poperechny, K. V., Maslova, S. V., Melchers, W. J. G., Bruins Slot, H. J. & Agol, V. I. ( 1996; ). Cis-element, oriR, involved in the initiation of (−) strand poliovirus RNA: a quasi-globular multi-domain RNA structure maintained by tertiary (‘kissing’) interactions. EMBO J 15, 5428–5436.
    [Google Scholar]
  33. Reed, L. J. & Muench, H. ( 1938; ). A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497.
    [Google Scholar]
  34. Rieder, E., Paul, A. V., Kim, D. W., van Boom, J. H. & Wimmer, E. ( 2000; ). Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation. J Virol 74, 10371–10380.[CrossRef]
    [Google Scholar]
  35. Thiviyanathan, V., Yang, Y., Kaluarachchi, K., Rijnbrand, R., Gorenstein, D. G. & Lemon, S. M. ( 2004; ). High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre). Proc Natl Acad Sci U S A 101, 12688–12693.[CrossRef]
    [Google Scholar]
  36. van Kuppeveld, F. J. M., Galama, J. M. D., Zoll, J. & Melchers, W. J. G. ( 1995; ). Genetic analysis of a hydrophobic domain of coxsackie B3 virus protein 2B: a moderate degree of hydrophobicity is required for a cis-acting function in viral RNA synthesis. J Virol 69, 7782–7790.
    [Google Scholar]
  37. van Kuppeveld, F. J. M., Hoenderop, J. G. J., Smeets, R. L. L., Willems, P. H. G. M., Dijkman, H. B. P. M., Galama, J. M. D. & Melchers, W. J. G. ( 1997; ). Coxsackievirus protein 2B modifies endoplasmic reticulum membrane and plasma membrane permeability and facilitates virus release. EMBO J 16, 3519–3532.[CrossRef]
    [Google Scholar]
  38. Wang, J., Bakkers, J. M. J. E., Galama, J. M. D., Bruins Slot, H. J., Pilipenko, E. V., Agol, V. I. & Melchers, W. J. G. ( 1999; ). Structural requirements of the higher order RNA kissing element in the enteroviral 3′UTR. Nucleic Acids Res 27, 485–490.[CrossRef]
    [Google Scholar]
  39. Wimmer, E., Hellen, C. U. T. & Cao, X. ( 1993; ). Genetics of poliovirus. Annu Rev Genet 27, 353–436.[CrossRef]
    [Google Scholar]
  40. Witwer, C., Rauscher, S., Hofacker, I. L. & Stadler, P. F. ( 2001; ). Conserved RNA secondary structures in Picornaviridae genomes. Nucleic Acids Res 29, 5079–5089.[CrossRef]
    [Google Scholar]
  41. Yang, Y., Rijnbrand, R., McKnight, K. L., Wimmer, E., Paul, A., Martin, A. & Lemon, S. M. ( 2002; ). Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14. J Virol 76, 7485–7494.[CrossRef]
    [Google Scholar]
  42. Yin, J., Paul, A. V., Wimmer, E. & Rieder, E. ( 2003; ). Functional dissection of a poliovirus cis-acting replication element [PV-cre(2C)]: analysis of single- and dual-cre viral genomes and proteins that bind specifically to PV-cre RNA. J Virol 77, 5152–5166.[CrossRef]
    [Google Scholar]
  43. Zuker, M., Mathews, D. H. & Turner, D. H. ( 1999; ). Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology, pp. 11–43. Edited by J. Barciszewski & B. F. C. Clark. Dordrecht: Kluwer.
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.81297-0
Loading
/content/journal/jgv/10.1099/vir.0.81297-0
Loading

Data & Media loading...

Supplements

vol. , part 1, pp. 103 – 113

Methods and references detailing plasmid construction [ PDF] (132 KB)



PDF

Most Cited This Month

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