Separation of primary structural components conferring autoregulation, transactivation, and DNA-binding properties to the herpes simplex virus transcriptional regulatory protein ICP4

Abstract

A truncated ICP4 peptide which contains the amino-terminal 774 amino acids of the 1,298-amino-acid polypeptide is proficient for DNA binding, autoregulation, and transactivation of some viral genes (N. A. DeLuca and P. A. Schaffer, J. Virol. 62:732-743, 1988) and hence exhibits many of the properties characteristic of intact ICP4. To define the primary sequence important for the activities inherent in the amino-terminal half of the ICP4 molecule, insertional and deletion mutagenesis of the sequences encoding these residues were conducted. The DNA-binding activity of the molecule as assayed by the association with a consensus binding site was sensitive to insertional mutagenesis in two closely linked regions of the molecule. One region between amino acids 445 and 487 is critical for DNA binding and may contain a helix-turn-helix motif. The second region between amino acids 263 and 338 reduces the binding activity to a consensus binding site. When analyzed in the viral background, the DNA-binding activity of a peptide containing an insertion at amino acid 338 to a consensus binding site was reduced while the association with an alternative sequence was eliminated, suggesting a possible mechanism by which ICP4 may recognize a broader range of sequence elements. Mutations which eliminated DNA binding also eliminated or reduced both transactivation and autoregulation, supporting the requirement for DNA binding for these activities. Peptides that retained the deduced DNA-binding domain but lacked amino acids 143 through 210 retained the ability to associate with the consensus site and autoregulatory activity but were deficient for transactivation, demonstrating that the structural requirements for transactivation are greater than those required for autoregulation.

Role of protein kinase A and the serine-rich region of herpes simplex virus type 1 ICP4 in viral replication

“…Previous studies have demonstrated that deletion of the conserved serine-rich sequence in the background of an ICP4 polypeptide truncated at residue 774 resulted in a loss of the ability to transactivate the early thymidine kinase gene promoter in transient assays (57). Paterson and Everett have also investigated the effect of mutations in the serine-rich region on the function of ICP4.…”

“…Clustered regions in the ICP4 primary structure are conserved among the ICP4 homologs of related alphaherpesviruses (4,21,37,60), suggesting the presence of functionally important domains. Genetic analysis of mutant ICP4 polypeptides has identified several such regions of conserved primary sequence that are required for the ICP4 regulatory activities (11,43,57). One of these regions, between amino acid residues 263 and 487, specifies the primary structure important for ICP4 DNA-binding activity (42,57).…”

“…Genetic analysis of mutant ICP4 polypeptides has identified several such regions of conserved primary sequence that are required for the ICP4 regulatory activities (11,43,57). One of these regions, between amino acid residues 263 and 487, specifies the primary structure important for ICP4 DNA-binding activity (42,57). This DNA-binding domain is clearly of functional importance, as both transactivation and repression mediated by ICP4 require its integrity, and most mutations which destroy the ability of ICP4 to bind to DNA render the protein nonfunctional or render the virus unable to grow (43,57).…”

“…One of these regions, between amino acid residues 263 and 487, specifies the primary structure important for ICP4 DNA-binding activity (42,57). This DNA-binding domain is clearly of functional importance, as both transactivation and repression mediated by ICP4 require its integrity, and most mutations which destroy the ability of ICP4 to bind to DNA render the protein nonfunctional or render the virus unable to grow (43,57). Another region, between amino acid residues 142 and 210 ( Fig.…”

“…This stretch, termed the serine-rich region, is well conserved among the sequenced alphaherpesviruses and is flanked by seven basic residues on the amino-terminal side and eight acidic residues on the carboxy-terminal side. The serine-rich region has been shown to be important for the induction of early and leaky late gene expression, particularly in mutants lacking the C-terminal sequences (42,57), and has been genetically implicated as a target for phosphorylation (11). Computer modeling of the serine-rich region predicts a loop structure (5,57).…”