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Journal of Virology, November 2007, p. 12272-12284, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.00984-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Crystallographic and Biochemical Analysis of Rotavirus NSP2 with Nucleotides Reveals a Nucleoside Diphosphate Kinase-Like Activity{triangledown}

Mukesh Kumar,1 Hariharan Jayaram,1,{dagger} Rodrigo Vasquez-Del Carpio,2,{ddagger} Xiaofang Jiang,1 Zenobia F. Taraporewala,2 Raymond H. Jacobson,3 John T. Patton,2 and B. V. Venkataram Prasad1*

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030,1 Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892,2 Department of Biochemistry and Molecular Biology, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 770303

Received 7 May 2007/ Accepted 20 August 2007

Rotavirus, the major pathogen of infantile gastroenteritis, carries a nonstructural protein, NSP2, essential for viroplasm formation and genome replication/packaging. In addition to RNA-binding and helix-destabilizing properties, NSP2 exhibits nucleoside triphosphatase activity. A conserved histidine (H225) functions as the catalytic residue for this enzymatic activity, and mutation of this residue abrogates genomic double-stranded RNA synthesis without affecting viroplasm formation. To understand the structural basis of the phosphatase activity of NSP2, we performed crystallographic analyses of native NSP2 and a functionally defective H225A mutant in the presence of nucleotides. These studies showed that nucleotides bind inside a cleft between the two domains of NSP2 in a region that exhibits structural similarity to ubiquitous cellular HIT (histidine triad) proteins. Only minor conformational alterations were observed in the cleft upon nucleotide binding and hydrolysis. This hydrolysis involved the formation of a stable phosphohistidine intermediate. These observations, reminiscent of cellular nucleoside diphosphate (NDP) kinases, prompted us to investigate whether NSP2 exhibits phosphoryl-transfer activity. Bioluminometric assay showed that NSP2 exhibits an NDP kinase-like activity that transfers the bound phosphate to NDPs. However, NSP2 is distinct from the highly conserved cellular NDP kinases in both its structure and catalytic mechanism, thus making NSP2 a potential target for antiviral drug design. With structural similarities to HIT proteins, which are not known to exhibit NDP kinase activity, NSP2 represents a unique example among structure-activity relationships. The newly observed phosphoryl-transfer activity of NSP2 may be utilized for homeostasis of nucleotide pools in viroplasms during genome replication.

* Corresponding author. Mailing address: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-5686. Fax: (713) 798-1625. E-mail: vprasad{at}bcm.tmc.edu

{triangledown} Published ahead of print on 5 September 2007.

{dagger} Present address: Howard Hughes Medical Institute and the Department of Biochemistry, Brandeis University, Waltham, MA 02454.

{ddagger} Present address: Structural Biology Program, Department of Molecular Physiology and Biophysics, Mount Sinai School of Medicine, New York, NY 10029.

Journal of Virology, November 2007, p. 12272-12284, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.00984-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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