The Unique IR2 Protein of Equine Herpesvirus 1 Negatively Regulates Viral Gene Expression

Abstract

The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dosedependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transienttransfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.The equine herpesvirus 1 (EHV-1) genome is comprised of 78 genes that are coordinately regulated and temporally expressed as immediate-early (IE), early (E), and late (L) ␥1 and true late ␥2 genes (10,16,17). The coordinated transcription of EHV-1 genes is regulated by six regulatory proteins that are expressed as one IE protein (IEP), four early proteins (EICP22P, EICP27P, EICP0P, and IR2P), and the late protein ETIF (5-7, 10, 13, 16-18, 20, 22, 24-26, 34-37). In addition, our ongoing work indicated that the IR3 transcript that is antisense to a portion of the IE mRNA negatively regulates IE gene expression (21; B. C. Ahn, S. K. Kim, and D. J. O'Callaghan, unpublished data).During a lytic infection, two transcripts arise from the IE open reading frame: a single, spliced 6.0-kb IE mRNA and a 3Ј-coterminal 4.4-kb early IR2 mRNA (20). The IEP (1,487 amino acids [aa]) is the major regulatory protein of EHV-1 and mediates the activation of transcription from E and some L viral promoters (34, 35) and is, thus, essential for replication (15). At the same time, the IEP downregulates its own promoter (34) and represses transcription of the true late glycoprotein K (gK) gene by binding to the transcription initiation site of the gK promoter (24). The IEP binds to the consensus binding sequence 5Ј-ATCGT-3Ј that overlaps the transcription initiation site of the IE promoter and to degenerate versions of this cognate...

Functional Characterization of the Serine-Rich Tract of Varicella-Zoster Virus IE62

“…In contrast, the IR2 protein by itself downregulated the ORF61 promoter (Fig. 4C, IR2P), which is consistent with our previous results (29,41). Plasmids pSV62TS2-IR2P and pSV62TS-IR2P, in which the 264-aa sequences between the TAD and SRT were partially and completely deleted, respectively, strongly trans-activated the ORF61 promoter (Fig.…”

“…6). The IR2 protein (IR2P), an early 1,165-aa truncated form of the EHV-1 IEP, lacks the TAD and SRT and negatively regulates viral gene expression (29,41). The validity of the chimeric fusion constructs was confirmed by DNA sequencing (data not shown).…”

“…The preparation of cytoplasmic and nuclear extracts of transfected cells and Western blot analysis were performed as previously described (41). Blots were incubated with a polyclonal antibody to IEP OC33 (42), a monoclonal antibody to ICP4 58S (ATCC), a polyclonal antibody to IE62 (sc-17525; Santa Cruz Biotechnology, Santa Cruz, CA), or a polyclonal antibody to green fluorescent protein (GFP) (sc-8334; Santa Cruz Biotech) for 2 h. The blots were washed three times for 10 min each in Tris-buffered saline plus Tween (TBST) and incubated with the secondary antibody (anti-rabbit IgG Fc-alkaline phosphatase [AP] conjugate [Promega]) for 1 h. Proteins were visualized by incubating the membranes containing blotted protein in AP conjugate substrate (AP conjugate substrate kit; Bio-Rad) according to the manufacturer's directions.…”

The α-TIF (VP16) Homologue (ETIF) of Equine Herpesvirus 1 Is Essential for Secondary Envelopment and Virus Egress

“…The sole IE gene of EHV-1, a homologue of the herpes simplex virus type 1 (HSV-1) ICP4 gene, is transcribed independently of de novo protein synthesis from gene 62 and is absolutely essential for virus replication (18). The EHV-1 IE protein is a multifunctional, regulatory protein capable of modulating early and late promoters independently of or synergistically with early regulatory proteins (4,25,27,55,56,67).IE gene transcription becomes augmented and strongly stimulated by the virion-associated transcriptional regulator of EHV-1 gene expression, variably referred to as ETIF or VP16-E, expressed from the true late (␥2) EHV-1 gene 12 (33,34,44,44,45,45). ETIF is the EHV-1 homologue of HSV-1 VP16 also known as ␣-trans-inducing factor (␣-TIF) (5).…”

“…The sole IE gene of EHV-1, a homologue of the herpes simplex virus type 1 (HSV-1) ICP4 gene, is transcribed independently of de novo protein synthesis from gene 62 and is absolutely essential for virus replication (18). The EHV-1 IE protein is a multifunctional, regulatory protein capable of modulating early and late promoters independently of or synergistically with early regulatory proteins (4,25,27,55,56,67).…”