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Journal of Virology, February 2006, p. 1807-1816, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1807-1816.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Hyperphosphorylation of the Rotavirus NSP5 Protein Is Independent of Serine 67 or NSP2, and the Intrinsic Insolubility of NSP5 Is Regulated by Cellular Phosphatases

Adrish Sen,1,2 Darin Agresti,1,2,3 and Erich R. Mackow1,2,3*

Department of Medicine,1 Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794,2 Northport VA Medical Center, Northport, New York 117683

Received 2 September 2005/ Accepted 17 November 2005

The NSP5 protein is required for viroplasm formation during rotavirus infection and is hyperphosphorylated into 32- to 35-kDa isoforms. Earlier studies reported that NSP5 is not hyperphosphorylated without NSP2 coexpression or deleting the NSP5 N terminus and that serine 67 is essential for NSP5 hyperphosphorylation. In this report, we show that full-length NSP5 is hyperphosphorylated in the absence of NSP2 or serine 67 and demonstrate that hyperphosphorylated NSP5 is predominantly present in previously unrecognized cellular fractions that are insoluble in 0.2% sodium dodecyl sulfate. The last 68 residues of NSP5 are sufficient to direct green fluorescent protein into insoluble fractions and cause green fluorescent protein localization into viroplasm-like structures; however, NSP5 insolubility was intrinsic and did not require NSP5 hyperphosphorylation. When we mutated serine 67 to alanine we found that the NSP5 mutant was both hyperphosphorylated and insoluble, identical to unmodified NSP5, and as a result serine 67 is not required for NSP5 phosphorylation. Interestingly, treating cells with the phosphatase inhibitor calyculin A permitted the accumulation of soluble hyperphosphorylated NSP5 isoforms. This suggests that soluble NSP5 is constitutively dephosphorylated by cellular phosphatases and demonstrates that hyperphosphorylation does not direct NSP5 insolubility. Collectively these findings indicate that NSP5 hyperphosphorylation and insolubility are completely independent parameters and that analyzing insoluble NSP5 is essential for studies assessing NSP5 phosphorylation. Our results also demonstrate the involvement of cellular phosphatases in regulating NSP5 phosphorylation and indicate that in the absence of other rotavirus proteins, domains on soluble and insoluble NSP5 recruit cellular kinases and phosphatases that coordinate NSP5 hyperphosphorylation.

* Corresponding author. Mailing address: Department of Medicine and Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794. Phone: (631) 444-2120. Fax: (631) 444-8886. E-mail: emackow{at}mail.som.sunysb.edu.

Journal of Virology, February 2006, p. 1807-1816, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1807-1816.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sen, A., Sen, N., Mackow, E. R. (2007). The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain. J. Virol. 81: 11758-11767 [Abstract] [Full Text]  
  • Campagna, M., Budini, M., Arnoldi, F., Desselberger, U., Allende, J. E., Burrone, O. R. (2007). Impaired hyperphosphorylation of rotavirus NSP5 in cells depleted of casein kinase 1{alpha} is associated with the formation of viroplasms with altered morphology and a moderate decrease in virus replication. J. Gen. Virol. 88: 2800-2810 [Abstract] [Full Text]  
  • Jiang, X., Jayaram, H., Kumar, M., Ludtke, S. J., Estes, M. K., Prasad, B. V. V. (2006). Cryoelectron Microscopy Structures of Rotavirus NSP2-NSP5 and NSP2-RNA Complexes: Implications for Genome Replication. J. Virol. 80: 10829-10835 [Abstract] [Full Text]  


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