Identification of a Motif in the C Terminus of Herpes Simplex Virus Regulatory Protein ICP4 That Contributes to Activation of Transcription

Abstract

Expression of most viral genes during productive infection by herpes simplex virus is regulated by the viral protein ICP4 (also called IE175 or Vmw175). The N-terminal portion of ICP4 contains well-defined transactivation, DNA binding, and dimerization domains that contribute to promoter regulation. The C-terminal half of ICP4 contributes to the activity of ICP4, but the functional motifs have not been well mapped. To localize functional motifs in the C-terminal half of ICP4, we have compared the relative specific activities of ICP4 variants in transient-transfection assays. Deletion of the C-terminal 56 residues reduces the specific activity more than 10-fold. Mutational analysis identified three consecutive residues (1252 to 1254) that are conserved in ICP4 orthologs and are essential for full activity, especially in the context of ICP4 variants with a deletion in the N-terminal transactivation domain. Recombinant viruses that encode variants of ICP4 with mutations in the N-terminal transactivation domain and/or the extreme C terminus were constructed. The phenotypes of these recombinant viruses support the hypothesis that efficient promoter activation by ICP4 requires motifs at both the N and C termini. The data suggest that the C terminus of ICP4 functions not as an independent transactivation domain but as an enhancer of the ICP4 N-terminal transactivation domain. The data provide further support for the hypothesis that some ICP4 motifs required for promoter activation are not required for promoter repression and suggest that ICP4 utilizes different cellular factors for activation or repression of viral promoters.During productive infection by herpes simplex virus type 1 (HSV-1), approximately 75 genes encoded within the linear 152-kbp viral genome are transcribed by RNA polymerase II in three sequential phases designated immediate early (IE or ␣), early (E or ␤), and late (L or ␥) (for a review, see reference 52). The immediate-early protein ICP4 (infected-cell polypeptide 4) is required for efficient transcription of early and late viral genes and thus is essential for productive infection. The immediate-early proteins ICP0, ICP22, and ICP27 enhance expression of early and late genes at both transcriptional and posttranscriptional levels and contribute to some functions of ICP4 (52).ICP4 is a 1,298-amino-acid (aa) phosphoprotein that binds DNA in a sequence-specific manner as a homodimer (14,16,17,34). ICP4 represses transcription from three viral genes (LAT, ICP4, and ORF-P) that have a high-affinity ICP4 binding site spanning the transcription initiation site (1,16,20,21,28,30,41,42). ICP4 stimulates transcription from early and late viral promoters through interactions with viral DNA and cellular proteins that are poorly understood. Although the ICP4 DNA binding domain is essential for activation of transcription (39, 45), extensive analyses have not revealed a specific sequence that binds ICP4 with high affinity and is common to all promoters activated by ICP4 (13, 52). The observation that the minima...

Genetic diversity of equine herpesvirus 1 isolated from neurological, abortigenic and respiratory disease outbreaks

“…ICP4, the sole immediate–early protein in EHV‐1, is essential for viral replication (Garko‐Buczynski, Smith, Kim, & O'Callaghan, 1998). There were four non‐synonymous changes in this region when compared to the consensus EHV‐1 sequence, but it is not known whether this has any functional outcome, although the C ‐terminus of HSV‐1 ICP4 is thought to enhance the functions of the transactivation domain located at the N ‐terminus (Bruce & Wilcox, 2002; Wagner, Bayer, & Deluca, 2013). A similar, but not identical, recombination event between EHV‐1 and EHV‐4 has been reported in the same ORF in an EHV‐1 strain isolated in Japan (Pagamjav, Sakata, Matsumura, Yamaguchi, & Fukushi, 2005).…”

Genome of Bovine Herpesvirus 5

“…Based on sequence homologies, alphaherpesvirus ICP4 orthologs are divided into regions I to V from the amino to the carboxy terminus (13,17,43,84). BHV-5 and BHV-1 ICP4 are most similar (90 to 100% amino acid identity) within regions II and IV.…”

DNA-Dependent Oligomerization of Herpes Simplex Virus Type 1 Regulatory Protein ICP4

“…The C-terminal regions of ICP4 analogs are highly conserved among different alphaherpesviruses (9,57). The available data indicate that even small mutations in the C-terminal domain of ICP4 significantly alter the biological activities of the protein (6,10). Studies are under way to determine the region within the C-terminal domain of ICP4 involved in DNA-dependent oligomerization to explore its contribution activation independent of other functions in the C-terminal region of ICP4.…”

“…ICP4 negatively regulates the ICP4 promoter (12, 41), latency-associated promoter (3), and L/ST promoter (5, 59) and activates most of the early and late genes of the virus (12,16,22,41,45,57,60). Genetic and biochemical analyses revealed that ICP4 contains discrete domains responsible for DNA binding, nuclear localization, and transcriptional regulation (6,14,43,44,49,51). ICP4 is a dimer in solution (39) and probably functions as a dimer in DNA binding and transcriptional regulation (40,50).…”