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Journal of Virology, May 2002, p. 5291-5299, Vol. 76, No. 10
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.10.5291-5299.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNA-Binding Activity of the Rotavirus Phosphoprotein NSP5 Includes Affinity for Double-Stranded RNA

Patrice Vende,1,2 Zenobia F. Taraporewala,1 and John T. Patton1*

Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,1 Laboratoire de Virologie et Immunologie Moléculaires INRA, CRJJ, Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France2

Received 14 November 2001/ Accepted 20 February 2002

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.

* Corresponding author. Mailing address: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Dr. - MSC 8026, Bethesda, MD 20892. Phone: (301) 594-1615. Fax: (301) 496-8312. E-mail: jpatton{at}niaid.nih.gov.

Journal of Virology, May 2002, p. 5291-5299, Vol. 76, No. 10
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.10.5291-5299.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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