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J. Biol. Chem., Vol. 280, Issue 12, 11955-11960, March 25, 2005

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Human Cytomegalovirus UL84 Is a Phosphoprotein That Exhibits UTPase Activity and Is a Putative Member of the DExD/H Box Family of Proteins^*

Kelly S. Colletti, Yiyang Xu, Irena Yamboliev, and Gregory S. Pari

From the University of Nevada-Reno, Department of Microbiology & Immunology and the Cell and Molecular Biology Program, Reno, Nevada 89557

Human cytomegalovirus (HCMV) UL84 is required for lytic DNA replication and is proposed to be the key factor in initiation of viral DNA synthesis. We now show that UL84 has a high degree of homology to the DExD/H (where x can be any amino acid) box family of helicases, displays UTPase activity, and is phosphorylated at serine residues. Affinity column-purified UL84-FLAG fusion protein was used in an in vitro nucleoside triphosphatase (NTPase) assay to show that UL84 has NTPase activity, preferring UTP. This UTPase activity was linear with respect to enzyme concentration and slightly enhanced by the addition of nucleic acid substrates. UL84 UTPase was the highest at low salt concentrations, a pH of 7.5, and a temperature of 45 °C. The enzyme preferred Mg²⁺ as the divalent cation but was also able to catalyze the UTPase reaction in the presence of Mn²⁺, Ca²⁺, and Zn²⁺ albeit at lower levels. The evidence presented here suggests that the UL84 UTPase activity may be part of an energy-generating system for helicase activity associated with the initiation of HCMV DNA replication.

Received for publication, December 21, 2004 , and in revised form, January 21, 2005.

^* This work was supported by National Institutes of Health Public Health Service Grant AI45096. 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.

To whom correspondence should be addressed: University of Nevada-Reno, Dept. of Microbiology, School of Medicine, Howard Bldg. Reno, NV 89557. Tel.: 775-784-4824; Fax: 775-327-2332; E-mail: gpari{at}med.unr.edu.

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