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Journal of Virology, November 2004, p. 11853-11864, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11853-11864.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Putative Zinc Finger of the Human Cytomegalovirus IE2 86-Kilodalton Protein Is Dispensable for DNA Binding and Autorepression, Thereby Demarcating a Concise Core Domain in the C Terminus of the Protein

Jasmin Asmar, Lüder Wiebusch, Matthias Truss, and Christian Hagemeier*

Laboratory for Molecular Biology, Department of Pediatrics, Charité, Humboldt-University, Berlin, Germany

Received 17 February 2004/ Accepted 28 June 2004

The IE2 86-kDa gene product is an essential regulatory protein of human cytomegalovirus (HCMV) with several functions, including transactivation, negative autoregulation, and cell cycle regulation. In order to understand the physiological significance of each of the IE2 functions, discriminating mutants of IE2 are required that can be tested in a viral background. However, no such mutants of IE2 are available, possibly reflecting structural peculiarities of the large and ill-defined C-terminal domain of IE2. Here, we revisited the C-terminal domain by analyzing IE2 mutants for transactivation, DNA binding, autoregulation, and cell cycle regulation in parallel. We found it to contain an unexpectedly concise core domain (amino acids 450 to 544) that is defined by its absolute sensitivity to any kind of mutation. In contrast, the region adjacent to the core (amino acids 290 to 449) generally tolerates mutations much better. Although it contributes more specific sequence information to distinct IE2 activities, none of the mutations analyzed abolished any particular function. The core is demarcated from the adjacent region by the putative zinc finger region (amino acids 428 to 452). Surprisingly, the deletion of the putative zinc finger region from IE2 revealed that this region is entirely dispensable for any of the IE2 functions tested here in transfection assays. Our work supports the view that the 100 amino acids of the core domain hold the key to most functions of IE2. A systematic, high-density mutational analysis of this region may identify informative mutants discriminating between various IE2 functions that can then be tested in a viral background.

* Corresponding author: Laboratory for Molecular Biology, Department of Pediatrics, Charité, Humboldt-University, Ziegelstr. 5-9, D-10098 Berlin, Germany. Phone: 49 30 450 566041. Fax: 49 30 450 566913. E-mail: christian.hagemeier{at}charite.de.

Journal of Virology, November 2004, p. 11853-11864, Vol. 78, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.21.11853-11864.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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