1887

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Volume 90, Issue 9

Functional differences between precursor and mature forms of the RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus Free

Abstract

The genome region encoding the RNA-dependent RNA polymerase 3CD-like precursor from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was cloned and expressed in using polyhistidine fusion-based vectors. The full-length recombinant 3CD-like precursor polypeptide could not be purified as a consequence of its autoproteolytic processing. A Cys→Gly substitution of the 3C-like catalytic cysteine (C1212) impeded the cleavage and allowed the purification of the precursor at high yields using a polyhistidine fusion expression vector. Equimolar amounts of purified recombinant precursor (C1212G mutant) and mature 3D-like polymerase showed significant activity differences in genome-linked protein (VPg) uridylylation and RNA polymerization using assays. The data indicated that the precursor was more active than the mature polymerase in catalysing RHDV VPg uridylylation, whereas the latter enzyme form had higher activity than its precursor in RNA polymerization assays using a heteropolymeric RNA template.

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2009-09-01
2024-04-20
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References

  1. Belliot, G., Sosnovtsev, S. V., Mitra, T., Hammer, C., Garfield, M. & Green, K. Y.(2003).In vitro proteolytic processing of the MD145 norovirus ORF1 nonstructural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells. J Virol 77, 10957–10974.[CrossRef] [Google Scholar]
  2. Belliot, G., Sosnovtsev, S. V., Chang, K. O., Babu, V., Uche, U., Arnold, J. J., Cameron, C. E. & Green, K. Y.(2005). Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase. J Virol 79, 2393–2403.[CrossRef] [Google Scholar]
  3. Belliot, G., Sosnovtsev, S. V., Chang, K. O., McPhie, P. & Green, K. Y.(2008). Nucleotidylylation of the VPg protein of a human norovirus by its proteinase–polymerase precursor protein. Virology 374, 33–49.[CrossRef] [Google Scholar]
  4. Boniotti, B., Wirblich, C., Sibilia, M., Meyers, G., Thiel, H. J. & Rossi, C.(1994). Identification and characterization of a 3C-like protease from rabbit hemorrhagic disease virus, a calicivirus. J Virol 68, 6487–6495. [Google Scholar]
  5. Green, K. Y., Ando, T., Balayan, M. S., Berke, T., Clarke, I. N., Estes, M. K., Matson, D. O., Nakata, S., Neill, J. D. & other authors(2000). Taxonomy of the caliciviruses. J Infect Dis 181, S322–S330.[CrossRef] [Google Scholar]
  6. Green, K. Y., Mory, A., Fogg, M. H., Weisberg, A., Belliot, G., Wagner, M., Mitra, T., Ehrenfeld, E., Cameron, C. E. & Sosnovtsev, S. V.(2002). Isolation of enzymatically active replication complexes from feline calicivirus-infected cells. J Virol 76, 8582–8595.[CrossRef] [Google Scholar]
  7. Konig, M., Thiel, H. J. & Meyers, G.(1998). Detection of viral proteins after infection of cultured hepatocytes with rabbit hemorrhagic disease virus. J Virol 72, 4492–4497. [Google Scholar]
  8. Liu, B. L., Viljoen, G. J., Clarke, I. N. & Lambden, P. R.(1999). Identification of further proteolytic cleavage sites in the Southampton calicivirus polyprotein by expression of the viral protease in E. coli. J Gen Virol 80, 291–296. [Google Scholar]
  9. López Vázquez, A., Martín Alonso, J. M., Casais, R., Boga, J. A. & Parra, F.(1998). Expression of enzymatically active rabbit hemorrhagic disease virus RNA-dependent RNA polymerase in Escherichia coli. J Virol 72, 2999–3004. [Google Scholar]
  10. López Vázquez, A., Martín Alonso, J. M. & Parra, F.(2000). Mutation analysis of the GDD sequence motif of a calicivirus RNA-dependent RNA polymerase. J Virol 74, 3888–3891.[CrossRef] [Google Scholar]
  11. López Vázquez, A., Martín Alonso, J. M. & Parra, F.(2001). Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli. Arch Virol 146, 59–69.[CrossRef] [Google Scholar]
  12. Machín, A., Martín Alonso, J. M. & Parra, F.(2001). Identification of the amino acid residue involved in rabbit hemorrhagic disease virus VPg uridylylation. J Biol Chem 276, 27787–27792.[CrossRef] [Google Scholar]
  13. Martín Alonso, J. M., Casais, R., Boga, J. A. & Parra, F.(1996). Processing of rabbit hemorrhagic disease virus polyprotein. J Virol 70, 1261–1265. [Google Scholar]
  14. Meyers, G., Wirblich, C. & Thiel, H. J.(1991). Rabbit hemorrhagic disease virus – molecular cloning and nucleotide sequencing of a calicivirus genome. Virology 184, 664–676.[CrossRef] [Google Scholar]
  15. Meyers, G., Wirblich, C., Thiel, H. J. & Thumfart, J. O.(2000). Rabbit hemorrhagic disease virus: genome organization and polyprotein processing of a calicivirus studied after transient expression of cDNA constructs. Virology 276, 349–363.[CrossRef] [Google Scholar]
  16. Ng, K. K., Cherney, M. M., Vazquez, A. L., Machin, A., Martín Alonso, J. M., Parra, F. & James, M. N.(2002). Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase. J Biol Chem 277, 1381–1387.[CrossRef] [Google Scholar]
  17. Ohlinger, V. F., Haas, B., Meyers, G., Weiland, F. & Thiel, H. J.(1990). Identification and characterization of the virus causing rabbit hemorrhagic disease. J Virol 64, 3331–3336. [Google Scholar]
  18. Oka, T., Katayama, K., Ogawa, S., Hansman, G. S., Kageyama, T., Ushijima, H., Miyamura, T. & Takeda, N.(2005). Proteolytic processing of sapovirus ORF1 polyprotein. J Virol 79, 7283–7290.[CrossRef] [Google Scholar]
  19. Parra, F. & Prieto, M.(1990). Purification and characterization of a calicivirus as the causative agent of a lethal hemorrhagic disease in rabbits. J Virol 64, 4013–4015. [Google Scholar]
  20. Pringle, C. R.(1998). Virus taxonomy – San Diego 1998. Arch Virol 143, 1449–1459.[CrossRef] [Google Scholar]
  21. Sosnovtseva, S. A., Sosnovtsev, S. V. & Green, K. Y.(1999). Mapping of the feline calicivirus proteinase responsible for autocatalytic processing of the nonstructural polyprotein and identification of a stable proteinase-polymerase precursor protein. J Virol 73, 6626–6633. [Google Scholar]
  22. Wei, L., Huhn, J. S., Mory, A., Pathak, H. B., Sosnovtsev, S. V., Green, K. Y. & Cameron, C. E.(2001). Proteinase-polymerase precursor as the active form of feline calicivirus RNA-dependent RNA polymerase. J Virol 75, 1211–1219.[CrossRef] [Google Scholar]
  23. Wirblich, C., Sibilia, M., Boniotti, M. B., Rossi, C., Thiel, H. J. & Meyers, G.(1995). 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the Orf1 polyprotein and analysis of cleavage specificity. J Virol 69, 7159–7168. [Google Scholar]
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Functional differences between precursor and mature forms of the RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus
J. Gen. Virol. 90, 2114 (2009); https://doi.org/10.1099/vir.0.011296-0
/content/journal/jgv/10.1099/vir.0.011296-0
/content/journal/jgv/10.1099/vir.0.011296-0
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