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. 2002 Feb;8(2):188–201. doi: 10.1017/s1355838202012785

Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C.

Roland Zell 1, Karim Sidigi 1, Enrico Bucci 1, Axel Stelzner 1, Matthias Görlach 1
PMCID: PMC1370242  PMID: 11911365

Abstract

The initiation of enteroviral positive-strand RNA synthesis requires the presence of a functional ribonucleoprotein complex containing a cloverleaf-like RNA secondary structure at the 5' end of the viral genome. Other components of the ribonucleoprotein complex are the viral 3CD proteinase (the precursor protein of the 3C proteinase and the 3D polymerase), the viral 3AB protein and the cellular poly(rC)-binding protein 2. For a molecular characterization of the RNA-binding properties of the enteroviral proteinase, the 3C proteinase of coxsackievirus B3 (CVB3) was bacterially expressed and purified. The recombinant protein is proteolytically active and forms a stable complex with in vitro-transcribed cloverleaf RNA of CVB3. The formation of stable complexes is also demonstrated with cloverleaf RNA of poliovirus (PV) 1, the first cloverleaf of bovine enterovirus (BEV) 1, and human rhinovirus (HRV) 2 but not with cloverleaf RNA of HRV14 and the second cloverleaf of BEV1. The apparent dissociation constants of the protein:RNA complexes range from approx. 1.7 to 4.6 microM. An electrophoretic mobility shift assay with subdomain D of the CVB3 cloverleaf demonstrates that this RNA is sufficient to bind the CVB3 3C proteinase. Binding assays using mutated versions of CVB3 and HRV14 cloverleaf RNAs suggest that the presence of structural features rather than a defined sequence motif of loop D are important for 3C proteinase-RNA interaction.

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Selected References

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

  1. Andino R., Rieckhof G. E., Achacoso P. L., Baltimore D. Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA. EMBO J. 1993 Sep;12(9):3587–3598. doi: 10.1002/j.1460-2075.1993.tb06032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andino R., Rieckhof G. E., Baltimore D. A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA. Cell. 1990 Oct 19;63(2):369–380. doi: 10.1016/0092-8674(90)90170-j. [DOI] [PubMed] [Google Scholar]
  3. Andino R., Rieckhof G. E., Trono D., Baltimore D. Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region. J Virol. 1990 Feb;64(2):607–612. doi: 10.1128/jvi.64.2.607-612.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blyn L. B., Swiderek K. M., Richards O., Stahl D. C., Semler B. L., Ehrenfeld E. Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11115–11120. doi: 10.1073/pnas.93.20.11115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borman A. M., Deliat F. G., Kean K. M. Sequences within the poliovirus internal ribosome entry segment control viral RNA synthesis. EMBO J. 1994 Jul 1;13(13):3149–3157. doi: 10.1002/j.1460-2075.1994.tb06613.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Böhm G., Muhr R., Jaenicke R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng. 1992 Apr;5(3):191–195. doi: 10.1093/protein/5.3.191. [DOI] [PubMed] [Google Scholar]
  7. Gamarnik A. V., Andino R. Two functional complexes formed by KH domain containing proteins with the 5' noncoding region of poliovirus RNA. RNA. 1997 Aug;3(8):882–892. [PMC free article] [PubMed] [Google Scholar]
  8. Grüne M., Görlach M., Soskic V., Klussmann S., Bald R., Fürste J. P., Erdmann V. A., Brown L. R. Initial analysis of 750 MHz NMR spectra of selective 15N-G,U labelled E. coli 5S rRNA. FEBS Lett. 1996 Apr 29;385(1-2):114–118. doi: 10.1016/0014-5793(96)00361-4. [DOI] [PubMed] [Google Scholar]
  9. Görlach M., Burd C. G., Dreyfuss G. The mRNA poly(A)-binding protein: localization, abundance, and RNA-binding specificity. Exp Cell Res. 1994 Apr;211(2):400–407. doi: 10.1006/excr.1994.1104. [DOI] [PubMed] [Google Scholar]
  10. Harris K. S., Xiang W., Alexander L., Lane W. S., Paul A. V., Wimmer E. Interaction of poliovirus polypeptide 3CDpro with the 5' and 3' termini of the poliovirus genome. Identification of viral and cellular cofactors needed for efficient binding. J Biol Chem. 1994 Oct 28;269(43):27004–27014. [PubMed] [Google Scholar]
  11. Huang H., Alexandrov A., Chen X., Barnes T. W., 3rd, Zhang H., Dutta K., Pascal S. M. Structure of an RNA hairpin from HRV-14. Biochemistry. 2001 Jul 10;40(27):8055–8064. doi: 10.1021/bi010572b. [DOI] [PubMed] [Google Scholar]
  12. Hämmerle T., Molla A., Wimmer E. Mutational analysis of the proposed FG loop of poliovirus proteinase 3C identifies amino acids that are necessary for 3CD cleavage and might be determinants of a function distinct from proteolytic activity. J Virol. 1992 Oct;66(10):6028–6034. doi: 10.1128/jvi.66.10.6028-6034.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Joachims M., Van Breugel P. C., Lloyd R. E. Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro. J Virol. 1999 Jan;73(1):718–727. doi: 10.1128/jvi.73.1.718-727.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnson K. H., Gray D. M., Morris P. A., Sutherland J. C. A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands. Biopolymers. 1990 Feb 5;29(2):325–333. doi: 10.1002/bip.360290205. [DOI] [PubMed] [Google Scholar]
  15. Johnson V. H., Semler B. L. Defined recombinants of poliovirus and coxsackievirus: sequence-specific deletions and functional substitutions in the 5'-noncoding regions of viral RNAs. Virology. 1988 Jan;162(1):47–57. doi: 10.1016/0042-6822(88)90393-5. [DOI] [PubMed] [Google Scholar]
  16. Kelly R. C., Jensen D. E., von Hippel P. H. DNA "melting" proteins. IV. Fluorescence measurements of binding parameters for bacteriophage T4 gene 32-protein to mono-, oligo-, and polynucleotides. J Biol Chem. 1976 Nov 25;251(22):7240–7250. [PubMed] [Google Scholar]
  17. Kusov Y. Y., Gauss-Müller V. In vitro RNA binding of the hepatitis A virus proteinase 3C (HAV 3Cpro) to secondary structure elements within the 5' terminus of the HAV genome. RNA. 1997 Mar;3(3):291–302. [PMC free article] [PubMed] [Google Scholar]
  18. Leong L. E., Walker P. A., Porter A. G. Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities. J Biol Chem. 1993 Dec 5;268(34):25735–25739. [PubMed] [Google Scholar]
  19. Lindberg A. M., Crowell R. L., Zell R., Kandolf R., Pettersson U. Mapping of the RD phenotype of the Nancy strain of coxsackievirus B3. Virus Res. 1992 Jul;24(2):187–196. doi: 10.1016/0168-1702(92)90006-u. [DOI] [PubMed] [Google Scholar]
  20. Matthews D. A., Dragovich P. S., Webber S. E., Fuhrman S. A., Patick A. K., Zalman L. S., Hendrickson T. F., Love R. A., Prins T. J., Marakovits J. T. Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11000–11007. doi: 10.1073/pnas.96.20.11000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mirmira S. R., Tinoco I., Jr A quadruple mutant T4 RNA hairpin with the same structure as the wild-type translational repressor. Biochemistry. 1996 Jun 18;35(24):7675–7683. doi: 10.1021/bi960415q. [DOI] [PubMed] [Google Scholar]
  22. Nicklin M. J., Harris K. S., Pallai P. V., Wimmer E. Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro. J Virol. 1988 Dec;62(12):4586–4593. doi: 10.1128/jvi.62.12.4586-4593.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Parsley T. B., Towner J. S., Blyn L. B., Ehrenfeld E., Semler B. L. Poly (rC) binding protein 2 forms a ternary complex with the 5'-terminal sequences of poliovirus RNA and the viral 3CD proteinase. RNA. 1997 Oct;3(10):1124–1134. [PMC free article] [PubMed] [Google Scholar]
  25. Ryan M. D., Flint M. Virus-encoded proteinases of the picornavirus super-group. J Gen Virol. 1997 Apr;78(Pt 4):699–723. doi: 10.1099/0022-1317-78-4-699. [DOI] [PubMed] [Google Scholar]
  26. Sprecher C. A., Johnson W. C., Jr Circular dichroism of the nucleic acid monomers. Biopolymers. 1977 Oct;16(10):2243–2264. doi: 10.1002/bip.1977.360161012. [DOI] [PubMed] [Google Scholar]
  27. Stoldt M., Wöhnert J., Görlach M., Brown L. R. The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases. EMBO J. 1998 Nov 2;17(21):6377–6384. doi: 10.1093/emboj/17.21.6377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Todd S., Towner J. S., Semler B. L. Translation and replication properties of the human rhinovirus genome in vivo and in vitro. Virology. 1997 Mar 3;229(1):90–97. doi: 10.1006/viro.1996.8416. [DOI] [PubMed] [Google Scholar]
  29. Walker P. A., Leong L. E., Porter A. G. Sequence and structural determinants of the interaction between the 5'-noncoding region of picornavirus RNA and rhinovirus protease 3C. J Biol Chem. 1995 Jun 16;270(24):14510–14516. doi: 10.1074/jbc.270.24.14510. [DOI] [PubMed] [Google Scholar]
  30. Wang Q. M., Johnson R. B. Activation of human rhinovirus-14 3C protease. Virology. 2001 Feb 1;280(1):80–86. doi: 10.1006/viro.2000.0760. [DOI] [PubMed] [Google Scholar]
  31. Xiang W., Harris K. S., Alexander L., Wimmer E. Interaction between the 5'-terminal cloverleaf and 3AB/3CDpro of poliovirus is essential for RNA replication. J Virol. 1995 Jun;69(6):3658–3667. doi: 10.1128/jvi.69.6.3658-3667.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zell R., Klingel K., Sauter M., Fortmüller U., Kandolf R. Coxsackieviral proteins functionally recognize the polioviral cloverleaf structure of the 5'-NTR of a chimeric enterovirus RNA: influence of species-specific host cell factors on virus growth. Virus Res. 1995 Dec;39(2-3):87–103. doi: 10.1016/0168-1702(95)00075-5. [DOI] [PubMed] [Google Scholar]
  33. Zell R., Sidigi K., Henke A., Schmidt-Brauns J., Hoey E., Martin S., Stelzner A. Functional features of the bovine enterovirus 5'-non-translated region. J Gen Virol. 1999 Sep;80(Pt 9):2299–2309. doi: 10.1099/0022-1317-80-9-2299. [DOI] [PubMed] [Google Scholar]
  34. Zell R., Stelzner A. Application of genome sequence information to the classification of bovine enteroviruses: the importance of 5'- and 3'-nontranslated regions. Virus Res. 1997 Oct;51(2):213–229. doi: 10.1016/s0168-1702(97)00096-8. [DOI] [PubMed] [Google Scholar]

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