Separation of requirements for protein-DNA complex assembly from those for functional activity in the herpes simplex virus regulatory protein Vmw65.
A transient expression system was developed which results in efficient synthesis of the regulatory protein Vmw65 of herpes simplex virus type 1 in eucaryotic cells. The gene for Vmw65 was linked to the cytomegalovirus immediate-early (IE) promoter-enhancer region in a plasmid containing the simian virus 40 origin of replication. When transfected into COS cells, Vmw65 was expressed from this vector in 25 to 50% of the cells, with total levels of the protein approaching 20% of those observed in infected cells. Vmw65 expressed in this system is functional for specific DNA-binding complex formation with the host cell octamer-binding protein TRF and for transactivation of IE gene expression. We therefore produced a series of carboxy-terminal truncated forms of Vmw65 to examine the structural requirements of the protein for these activities. Deletion of the acidic carboxy-terminal 56 amino acids had no effect on DNA-binding complex formation but completely abolished the ability to transactivate. Amino acids between residues 434 and 453, a region which exhibits a high negative charge, were critical for IE transactivation. In contrast, the requirements for complex formation are located entirely within the N-terminal 403 amino acids, and our results indicate a requirement for this activity for residues between 316 and 403. Together with our previous work, the results presented here indicate that recruitment of TRF into a specific DNA-binding complex on IE consensus signals is required but not sufficient for functional IE transactivation by Vmw65.
Greaves
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R
Marie Curie Research Institute, Surrey, United Kingdom.
O'Hare
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P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D046911:Macromolecular Substances; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D018139:Simplexvirus / Q000502:physiology*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000502:physiology*; D014162:Transfection
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D014157:Transcription Factors