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Journal of Virology, November 2007, p. 11758-11767, Vol. 81, No. 21
0022-538X/07/$08.00+0     doi:10.1128/JVI.01124-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Formation of Viroplasm-Like Structures by the Rotavirus NSP5 Protein Is Calcium Regulated and Directed by a C-Terminal Helical Domain{triangledown}

Adrish Sen,1,2 Nandini Sen,1,2 and Erich R. Mackow1,2,3,4*

Department of Medicine,1 Department of Molecular Genetics and Microbiology,2 Molecular and Cellular Biology Graduate Program, SUNY at Stony Brook, Stony Brook, New York 11794,3 Northport VA Medical Center, Northport, New York 117684

Received 23 May 2007/ Accepted 7 August 2007

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic {alpha}-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 {alpha}-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted {alpha}-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.

* Corresponding author. Mailing address: Departments of Medicine and Molecular Genetics and Microbiology, HSC T17, Rm. 60, SUNY at Stony Brook, Stony Brook, NY 11794. Phone: (631) 444-2120. Fax: (631) 444-8886. E-mail: Erich.Mackow{at}stonybrook.edu

{triangledown} Published ahead of print on 15 August 2007.

Journal of Virology, November 2007, p. 11758-11767, Vol. 81, No. 21
0022-538X/07/$08.00+0     doi:10.1128/JVI.01124-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zambrano, J. L., Diaz, Y., Pena, F., Vizzi, E., Ruiz, M.-C., Michelangeli, F., Liprandi, F., Ludert, J. E. (2008). Silencing of Rotavirus NSP4 or VP7 Expression Reduces Alterations in Ca2+ Homeostasis Induced by Infection of Cultured Cells. J. Virol. 82: 5815-5824 [Abstract] [Full Text]  


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