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Journal of Virology, July 2007, p. 7077-7085, Vol. 81, No. 13
0022-538X/07/$08.00+0     doi:10.1128/JVI.00058-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus UL84 Interacts with an RNA Stem-Loop Sequence Found within the RNA/DNA Hybrid Region of oriLyt{triangledown}

Kelly S. Colletti,1 Kate E. Smallenburg,1 Yiyang Xu,2 and Gregory S. Pari1*

University of Nevada-Reno, School of Medicine, Department of Microbiology & Immunology and the Cell and Molecular Biology Program, Reno, Nevada 89557,1 G. W. Hooper Foundation, Box 0552, University of California, San Francisco, San Francisco, California 94143-05522

Received 9 January 2007/ Accepted 18 April 2007

Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex cis-acting oriLyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within oriLyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to oriLyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within oriLyt.

* Corresponding author. Mailing address: University of Nevada—Reno, Department of Microbiology, School of Medicine, Howard Bldg., Reno, NV 89557. Phone: (775) 784-4824. Fax: (775) 327-2332. E-mail: gpari{at}medicine.nevada.edu

{triangledown} Published ahead of print on 25 April 2007.

Journal of Virology, July 2007, p. 7077-7085, Vol. 81, No. 13
0022-538X/07/$08.00+0     doi:10.1128/JVI.00058-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kaiser, N., Lischka, P., Wagenknecht, N., Stamminger, T. (2009). Inhibition of Human Cytomegalovirus Replication via Peptide Aptamers Directed against the Nonconventional Nuclear Localization Signal of the Essential Viral Replication Factor pUL84. J. Virol. 83: 11902-11913 [Abstract] [Full Text]  
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