The infected cell polypeptide 4 (ICP4) of herpes simplex virus 1 (HSV-1) is a regulator of viral transcription that is required for productive infection. Since viral genes are transcribed by cellular RNA polymerase II (RNA pol II), ICP4 must interact with components of the pol II machinery to regulate viral gene expression. It has been shown previously that ICP4 interacts with TATA box-binding protein (TBP), TFIIB, and the TBPassociated factor 1 (TAF1) in vitro. In this study, ICP4-containing complexes were isolated from infected cells by tandem affinity purification (TAP). Forty-six proteins that copurified with ICP4 were identified by mass spectrometry. Additional copurifying proteins were identified by Western blot analysis. These included 11 components of TFIID and 4 components of the Mediator complex. The significance of the ICP4-Mediator interaction was further investigated using immunofluorescence and chromatin immunoprecipitation. Mediator was found to colocalize with ICP4 starting at early and continuing into late times of infection. In addition, Mediator was recruited to viral promoters in an ICP4-dependent manner. Taken together, the data suggest that ICP4 interacts with components of TFIID and Mediator in the context of viral infection, and this may explain the broad transactivation properties of ICP4.During lytic infection, herpes simplex virus 1 (HSV-1) exhibits a strictly regulated temporal cascade of gene expression that is divided into three general stages, immediate early (IE) or ␣, early (E) or ␤, and late (L) or ␥ (43, 44). This expression program is a result of a complex interplay between viral and cellular factors at both the transcriptional and posttranscriptional levels. HSV genes are transcribed by cellular RNA polymerase II (RNA pol II) (1, 14). The IE protein is required for productive infection (15,21,75). ICP4 activates the transcription of most viral genes while repressing the transcription of several viral genes, including its own (16, 27, 34, 35,66,67,74,79,103).ICP4 exists as a 350-kDa homodimeric (59, 90) phosphoprotein (72). It has a DNA binding/dimerization domain and nuclear localization signal that are flanked by regions involved in transactivation and repression (17,18, 32,70,71,89). ICP4 binds to the consensus DNA sequence ATCGTCNNNNYC GRC, where R is purine, Y is pyrimidine, and N is any base (19, 28), and it also has been shown to bind DNA nonspecifically (31). The binding of ICP4 to specific sites has been shown to be involved in the repression of the ICP4, LAT, and OrfP/ L/ST promoters (29, 38,52,62,83). While DNA binding appears to be necessary, no specific ICP4 binding sites have been unambiguously associated with activation (12,23, 26, 41,92).RNA pol II transcription is initiated through the assembly of the preinitiation complex (PIC) consisting of the general transcription factors (GTFs) (TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH) and RNA pol II (7,61,68). The efficiency of the formation of the PIC is a critical step in determining the rate of transcription and ...

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.

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