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J. Biol. Chem., Vol. 279, Issue 11, 10624-10633, March 12, 2004

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Role of the Histidine Triad-like Motif in Nucleotide Hydrolysis by the Rotavirus RNA-packaging Protein NSP2^*

Rodrigo Vasquez-Del Carpio¶, Fernando D. González-Nilo||, Hariharan Jayaram**, Eugenio Spencer, B. V. Venkataram Prasad**, John T. Patton, and Zenobia F. Taraporewala

From the Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892, the Laboratorio de Virologia and the ^||Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40 Correo 33, Santiago, Chile, and the ^**Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030

Octamers formed by the nonstructural protein NSP2 of rotavirus are proposed to function as molecular motors in the packaging of the segmented double-stranded RNA genome. The octamers have RNA binding, helix unwinding, and Mg²⁺-dependent NTPase activities and play a crucial role in assembly of viral replication factories (viroplasms). Comparison of x-ray structures has revealed significant structural homology between NSP2 and the histidine triad (HIT) family of nucleotidyl hydrolases, which in turn has suggested the location of the active site for NTP hydrolysis in NSP2. Consistent with the structural predictions, we show here using site-specific mutagenesis and ATP docking simulations that the active site for NTP hydrolysis is localized to residues within a 25-Å-deep cleft between the C- and N-terminal domains of the NSP2 monomer. Although lacking the precise signature HIT motif (HØHØHØØ where Ø is a hydrophobic residue), our analyses demonstrate that histidines (His²²¹ and His²²⁵) represent critical residues of the active site. Similar to events occurring during nucleotide hydrolysis by HIT proteins, NTP hydrolysis by NSP2 was found to produce a short lived phosphorylated intermediate. Evaluation of the biological importance of the NTPase activity of NSP2 by transient expression in mammalian cells showed that such activity has no impact on the ability of NSP2 to induce the hyperphosphorylation of NSP5 or to interact with NSP5 to form viroplasm-like structures. Hence the NTPase activity of NSP2 probably has a role subsequent to the formation of viroplasms, consistent with its suspected involvement in RNA packaging and/or replication.

Received for publication, October 22, 2003 , and in revised form, December 16, 2003.

^* This work was supported in part by a grant from the Fondo Nacional de Desarrollo Científico y Tecnológico, Chile (to E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by fellowships from the German Academic Exchange Service (Deutscher Akademischer Austausch Dienst) and from the Comisión Nacional de Investigación Científica y Tecnológica, Chile.

Supported by NIH Grant AI36040.

To whom correspondence should be addressed: Laboratory of Infectious Diseases, NIAID, National Institutes of Health, 50 South Dr., MSC 8026, Rm. 6314, Bethesda, MD 20892. Tel.: 301-594-1663; Fax: 301-496-8312; E-mail: ztarapore{at}niaid.nih.gov.

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