Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays

Gene
J H AhnG S Hayward

Abstract

The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region. Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so. Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells. In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2. Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas...Continue Reading

References

Apr 1, 1981·Proceedings of the National Academy of Sciences of the United States of America·L Guarente, M Ptashne
Dec 1, 1995·Journal of Virology·H ZhuT Shenk

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Citations

Mar 1, 2008·Molecular Immunology·James A ListmanPhilip E Auron
Nov 21, 2007·Reviews in Medical Virology·Beatrice MercorelliGiorgio Palù
Jan 17, 2009·Cell Division·Adam J Hume, Robert F Kalejta
Aug 11, 2017·Journal of Virology·Mitsuhiro NishimuraYasuko Mori
Jun 8, 2014·Journal of Neurology, Neurosurgery, and Psychiatry·Mahua DeyMaciej S Lesniak
Dec 21, 2019·The Journal of General Virology·Fei YuLiqun Lu

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