The N Terminus and C Terminus of Herpes Simplex Virus 1 ICP4 Cooperate To Activate Viral Gene Expression

Abstract

Infected cell polypeptide 4 (ICP4) activates transcription from most viral promoters. Two transactivation domains, one N-terminal and one C terminal, are largely responsible for the activation functions of ICP4. A mutant ICP4 molecule lacking the C-terminal activation domain (n208) efficiently activates many early genes, whereas late genes are poorly activated, and virus growth is severely impaired. The regions within the N terminus of ICP4 (amino acids 1 to 210) that contribute to activation were investigated by analysis of deletion mutants in the presence or absence of the C-terminal activation domain. The mutants were assessed for their abilities to support viral replication and to regulate gene expression. Several deletions in regions conserved in other alphaherpesviruses resulted in impaired activation and viral growth, without affecting DNA binding. The single small deletion that had the greatest effect on activation in the absence of the C terminus corresponded to a highly conserved stretch of amino acids between 81 and 96, rendering the molecule nonfunctional. However, when the C terminus was present, the same deletion had a minimal effect on activity. The amino terminus of ICP4 was predicted to be relatively disordered compared to the DNA-binding domain and the C-terminal 500 amino acids. Moreover, the amino terminus appears to be in a relatively extended conformation as determined by the hydrodynamic properties of several mutants. The data support a model where the amino terminus is an extended and possibly flexible region of the protein, allowing it to efficiently interact with multiple transcription factors at a distance from where it is bound to DNA, thereby enabling ICP4 to function as a general activator of polymerase II transcription. The C terminus of ICP4 can compensate for some of the mutations in the N terminus, suggesting that it either specifies redundant interactions or enables the amino terminus to function more efficiently.

Requirement of the N-Terminal Activation Domain of Herpes Simplex Virus ICP4 for Viral Gene Expression

“…1A). Deletion of the entire region between amino acids 30 and 210 completely eliminated activation, but this was only observed in the context of a molecule truncated at amino acid 774 (23). Repeated attempts to construct a virus with amino acids 30 to 210 deleted in the otherwise intact ICP4 gene were unsuccessful, possibly because the deletion interferes with the action of wt ICP4 provided by the complementing cells.…”

“…In a previous study, we showed that a number of deletions within a region between amino acids 30 and 210 had an effect on the ability of ICP4 to activate transcription, but none completely abolished activation in the context of the otherwise intact ICP4 molecule (23). This region comprises the majority of the aminoterminal transactivation domain without interfering with the DNA binding domain (7,8,15) (Fig.…”

“…However, late gene expression and viral growth are impaired (15). The deletion of amino acids 30 to 210 in the absence of the C-terminal 520 amino acids completely abolishes activation and viral growth (23). This study further investigates the requirement and activities of the N-terminal transactivation domain with respect to viral gene transcription and the possible sufficiency of the C-terminal transactivation domain.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses were performed as previously described (23). Briefly, 10% polyacrylamide Tris-HCl gels (Bio-Rad) were loaded with samples diluted in Laemmli buffer (0.05 M Tris-HCl, 2% SDS, 5% ␤-mercaptoethanol, 0.01% bromophenol blue, 5% sucrose).…”

“…Metabolic labeling experiments were performed as previously described (23,24) with the following modifications. A total of 5 ϫ 10 5 Vero, E5, or D14 cells were infected with d120 at a multiplicity of infection (MOI) of 10 PFU/cell for 1 h at room temperature.…”