Skip to content
1887

Journal of General Virology header logo Journal of General Virology

Volume 91, Issue 7

Murine norovirus-1 3D exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg No Access

Abstract

We investigated the roles and biochemical properties of recombinant murine norovirus-1 (MNV-1) 3D in RNA synthesis and virus genome-linked protein (VPg) nucleotidylylation. We therefore expressed VPg and 3D of MNV-1 in . MNV-1 3D exhibited RNA-dependent RNA polymerase (RdRp) activity with poly(A) RNA as a template and MnCl as a cofactor. MNV-1 3D demonstrated optimum RNA-synthesis activity at pH 7.4 and 37 °C in the absence of a primer. Further, VPg was guanylylated by MNV-1 3D in the presence of MnCl in a template-independent manner. The guanylylation reaction conducted with VPg substitution mutants (Y26F, Y40F, Y45F and Y117F) and a deletion mutant (Δ117–124) indicated that Tyr was the probable target site of guanylylation. Homopolymeric RNAs did not enhance VPg guanylylation, whereas -transcribed (−) subgenomic (SG) and (+)SG RNA enhanced VPg guanylylation by 9.2 and 3.2 times, respectively. Within (−)SG RNA, the (−)ORF3 region played a critical role in enhancing VPg guanylylation, suggesting that the MNV-1 ORF3 region of negative-strand RNA contains a -acting element that stimulates 3D-mediated VPg guanylylation.

  • Received:
  • Accepted:
  • Published Online:
© SGM
Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.020461-0
2010-07-01
2025-05-15
Loading full text...

Full text loading...

References

  1. Ambros, V. & Baltmore, D.(1978). Protein is linked to the 5′ end of poliovirus RNA by a phosphodiester linkage to tyrosine. J Biol Chem 253, 5263–5266. [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. Chaudhry, Y., Nayak, A., Bordeleau, M. E., Tanaka, J., Pelletier, J., Belsham, G. J., Roberts, L. O. & Goodfellow, I. G.(2006). Caliciviruses differ in their functional requirements for eIF4F components. J Biol Chem 281, 25315–25325.[CrossRef] [Google Scholar]
  5. Crotty, S., Gohara, D., Gilligan, D. K., Karelsky, S., Cameron, C. E. & Andino, R.(2003). Manganese-dependent polioviruses caused by mutations within the viral polymerase. J Virol 77, 5378–5388.[CrossRef] [Google Scholar]
  6. Daughenbaugh, K. F., Fraser, C. S., Hershey, J. W. & Hardy, M. E.(2003). The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment. EMBO J 22, 2852–2859.[CrossRef] [Google Scholar]
  7. Daughenbaugh, K. F., Wobus, C. E. & Hardy, M. E.(2006). VPg of murine norovirus binds translation initiation factors in infected cells. Virol J 3, 33[CrossRef] [Google Scholar]
  8. Fukushi, S., Kojima, S., Takai, R., Hoshino, F. B., Oka, T., Takeda, N., Katayama, K. & Kageyama, T.(2004). Poly(A)- and primer-independent RNA polymerase of Norovirus. J Virol 78, 3889–3896.[CrossRef] [Google Scholar]
  9. Fullerton, S. W. B., Blaschke, M., Coutard, B., Gebhardt, J., Gorbalenya, A., Canard, B., Tucker, P. A. & Rohayem, J.(2007). Structural and functional characterization of sapovirus RNA-dependent RNA polymerase. J Virol 81, 1858–1871.[CrossRef] [Google Scholar]
  10. 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]
  11. Goodfellow, I., Chaudhry, Y., Gioldasi, I., Gerondopoulos, A., Natoni, A., Labrie, L., Laliberte, J. F. & Roberts, L.(2005). Calicivirus translation initiation requires an interaction between VPg and eIF4E. EMBO Rep 6, 968–972.[CrossRef] [Google Scholar]
  12. Green, K. Y.(2007). Caliciviruses: the noroviruses. In Fields Virology, 5th edn, pp. 949–979. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
  13. Hsu, C. C., Wobus, C. E., Steffen, E. K., Riley, L. K. & Livingston, R. S.(2005). Development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase PCR assay to detect murine norovirus 1 infection in mice. Clin Diagn Lab Immunol 12, 1145–1151. [Google Scholar]
  14. Karst, S. M., Wobus, C. E., Lay, M., Davidson, J. & Virgin, H. W.(2003). STAT1-dependent innate immunity to a Norwalk-like virus. Science 299, 1575–1578.[CrossRef] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. Machín, A., Martín Alonso, J. M., Dalton, K. P. & Parra, F.(2009). Functional differences between precursor and mature forms of the RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus. J Gen Virol 90, 2114–2118.[CrossRef] [Google Scholar]
  20. Mitra, T., Sosnovtsev, S. V. & Green, K. Y.(2004). Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus. J Virol 78, 4931–4935.[CrossRef] [Google Scholar]
  21. Morales, M., Bárcena, J., Ramírez, M. A., Boga, J. A., Parra, F. & Torres, J. M.(2004). Synthesis in vitro of rabbit hemorrhagic disease virus subgenomic RNA by internal initiation on (−)sense genomic RNA: mapping of a subgenomic promoter. J Biol Chem 279, 17013–17018.[CrossRef] [Google Scholar]
  22. 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]
  23. Murphy, J. F., Rychlik, W., Rhoads, R. E., Hunt, A. G. & Shaw, J. G.(1991). A tyrosine residue in the small nuclear inclusion protein of tobacco vein mottling virus links the VPg to the viral RNA. J Virol 65, 511–513. [Google Scholar]
  24. Murphy, J. F., Klein, P. G., Hunt, A. G. & Shaw, J. G.(1996). Replacement of the tyrosine residue that links a potyviral VPg to the viral RNA is lethal. Virology 220, 535–538.[CrossRef] [Google Scholar]
  25. 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]
  26. Ng, K. K. S., Pendas-Franco, N., Rojo, J., Boga, J. A., Machín, A., Martín Alonso, J. M. & Parra, F.(2004). Crystal structure of Norwalk virus polymerase reveals the carboxyl terminus in the active site cleft. J Biol Chem 279, 16638–16645.[CrossRef] [Google Scholar]
  27. 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]
  28. 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]
  29. Puustinen, P. & Mäkinen, K.(2004). Uridylylation of the potyvirus VPg by viral replicase NIb correlates with the nucleotide binding capacity of VPg. J Biol Chem 279, 38103–38110.[CrossRef] [Google Scholar]
  30. Ranjith-Kumar, C. T., Kim, Y. C., Gutshall, L., Silverman, C., Khandekar, S., Sarisky, R. T. & Kao, C. C.(2002). Mechanism of de novo initiation by the hepatitis C virus RNA-dependent RNA polymerase: role of divalent metals. J Virol 76, 12513–12525.[CrossRef] [Google Scholar]
  31. Richards, O. C., Spagnolo, J. F., Lyle, J. M., Vleck, S. E., Kuchta, R. D. & Kirkegaard, K.(2006). Intramolecular and intermolecular uridylylation by poliovirus RNA-dependent RNA polymerase. J Virol 80, 7405–7415.[CrossRef] [Google Scholar]
  32. 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]
  33. Rohayem, J., Jager, K., Robel, I., Scheffler, U., Temme, A. & Rudolph, W.(2006a). Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis. J Gen Virol 87, 2621–2630.[CrossRef] [Google Scholar]
  34. Rohayem, J., Robel, I., Jager, K., Scheffler, U. & Rudolph, W.(2006b). Protein-primed and de novo initiation of RNA synthesis by norovirus 3Dpol. J Virol 80, 7060–7069.[CrossRef] [Google Scholar]
  35. Rothberg, P. G., Harris, T. J. R., Nomoto, A. & Wimmer, E.(1978).O4-(5′-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus. Proc Natl Acad Sci U S A 75, 4868–4872.[CrossRef] [Google Scholar]
  36. Sosnovtsev, S. V., Belliot, G., Chang, K. O., Prikhodko, V. G., Thackray, L. B., Wobus, C. E., Karst, S. M., Virgin, H. W. & Green, K. Y.(2006). Cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells. J Virol 80, 7816–7831.[CrossRef] [Google Scholar]
  37. van Ooij, M. J. M., Vogt, D. A., Paul, A., Castro, C., Kuijpers, J., van Kuppeveld, F. J. M., Cameron, C. E., Wimmer, E., Andino, R. & Melchers, W. J. G.(2006). Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis. J Gen Virol 87, 103–113.[CrossRef] [Google Scholar]
  38. 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]
  39. Wobus, C. E., Karst, S. M., Thackray, L. B., Chang, K. O., Sosnovtsev, S. V., Belliot, G., Krug, A., Mackenzie, J. M., Green, K. Y. & Virgin, H. W., IV(2004). Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2, e432[CrossRef] [Google Scholar]
  40. Wobus, C. E., Thackray, L. B. & Virgin, H. W., IV(2006). Murine norovirus: a model system to study norovirus biology and pathogenesis. J Virol 80, 5104–5112.[CrossRef] [Google Scholar]
/content/journal/jgv/10.1099/vir.0.020461-0
Loading
Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg
J. Gen. Virol. 91, 1713 (2010); https://doi.org/10.1099/vir.0.020461-0
/content/journal/jgv/10.1099/vir.0.020461-0
/content/journal/jgv/10.1099/vir.0.020461-0
Loading

Data & Media loading...

Supplements

vol. , part 7, pp. 1713–1722

Primers used in this study [ PDF] (75 KB)

PDF

Most cited Most Cited RSS feed

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