Mapping the Sequences That Mediate Interaction of the Equine Herpesvirus 1 Immediate-Early Protein and Human TFIIB

Abstract

Among the eight equid herpesviruses identified to date (52), equine herpesvirus 1 (EHV-1) is one of the most pathogenic herpesviruses of horses, causing spontaneous abortions in pregnant mares, as well as respiratory tract infections and neurological disorders (1,12,45). The virus is a member of the subfamily Alphaherpesvirinae and serves as a model for the investigation of alphaherpesvirus gene regulation during both productive and persistent infections. The 77 EHV-1 genes are temporally and coordinately expressed at immediate-early (IE), early, and late (␥1 and ␥2) times of the lytic infection cycle (8, 18), analogous to that of herpes simplex virus type 1 (HSV-1) (11,33). In contrast to HSV-1, EHV-1 carries only one IE gene (also termed IR1 gene) that is expressed without prior viral protein synthesis due to the EHV-1 ␣-trans-inducing factor (ETIF), a homolog of the HSV-1 VP16 protein (14,41,47). The EHV-1 IE gene (i) is located within each invertedrepeat region and encodes a polypeptide of 1,487 amino acids (aa) with a predicted molecular mass of approximately 155 kDa (19,21,27), (ii) has a product with a high degree of homology with HSV-1 ICP4 and the varicella-zoster virus ORF62 gene products (21), and (iii) is transcribed as a 6.0-kb spliced mRNA (19,27,51) that gives rise to both structurally and antigenically related protein species ranging from 125 to 200 kDa (7,8,51). In transient-cotransfection assays, the IE protein is a bifunctional regulatory protein capable of (i) negatively autoregulating its own promoter (55), (ii) independently activating EHV-1 early and heterologous viral promoters (55, 56), (iii) cooperating synergistically with two early auxiliary regulatory proteins (EICP22 and EICP27) to activate EHV-1 early and ␥1 late promoters (32,44,55,57,64), and (iv) acting antagonistically with a third early major regulatory protein, EICP0, to selectively repress expression of certain promoters from all classes of EHV-1 promoters, including ␥2 late promoters (3,35).Sequence alignment of the EHV-1 IE protein and other homologs in the subfamily Alphaherpesvirinae defined five colinear regions that harbor specific functional domains. Region 1 contains an acidic transactivation domain (TAD; aa 3 to 89) (58) and a serine-rich tract (SRT; aa 181 to 220). Regions 2 and 3 harbor a helix-loop-helix motif that mediates a sequencespecific DNA-binding activity (aa 422 to 597) (38), while the nuclear localization signal (aa 963 to 970) lies within region 3 (56). Region 5 contains a transcriptional-enhancement domain that is required for the full transactivation activity of the IE protein (5, 56

A Negative Regulatory Element (Base Pairs −204 to −177) of the EICP0 Promoter of Equine Herpesvirus 1 Abolishes the EICP0 Protein's trans -Activation of Its Own Promoter

“…3). Also, the IE protein binds to factors in the basal transcriptional machinery such as TFIIB and TBP (1,2,29) and in addition binds to other EHV-1 regulatory proteins (11,12,35).…”

Interaction of the Equine Herpesvirus 1 EICP0 Protein with the Immediate-Early (IE) Protein, TFIIB, and TBP May Mediate the Antagonism between the IE and EICP0 Proteins

“…8A). Plasmid pGSTKG-TFIIB, which contains the entire TFIIB coding sequence cloned in frame with the GST gene and expresses the fusion protein GST-hIIB (1-316), was described previously (30). To generate plasmid pGST4T1-TFIIB, pGBKTFIIB was digested with SmaI and SalI, and the released TFIIB gene was cloned into the SmaI and SalI sites of the pGEX-4T1 (Promega).…”

“…Plasmids pGST-IE(1-1487), pGST-IE(407-539), pGST-IE(422-575), and pGST-IE(898-1487), which express IE aa 1 to 1487, 407 to 539, 422 to 575, and 898 to 1487 as glutathione S-transferase (GST) fusion proteins, respectively, have been described previously (36). Plasmids pGST-IE(1-960), pGST-IE(1-424), and pGST-IE(1-289), pGST-IE (1-88), pGST-IE(179-424), pGST-IE(539-910), and pGST-IE(323-1487) have been described previously (30).…”

“…The HSV-1 ICP4 forms a tripartite complex with TFIIB and TBP (55) and promotes PIC formation by enhancing the binding of TFIID to the TATA box element (20). Our previous results have shown that the interaction of the EHV-1 IE protein with TFIIB is necessary for its full trans-activation function and that the IE-TFIIB interaction may be part of the mechanism by which the IE protein activates transcription (30). The large T antigen of simian virus 40 (SV40) (31), the E1A protein of adenovirus (17), and the EBNA2 and ZEBRA proteins of Epstein-Barr virus (8,43) are other examples of viral trans-activators that directly interact with TFIID and/or TFIIB.…”

Equine herpesvirus type 1 replication is delayed in CD172a+ monocytic cells and controlled by histone deacetylases

“…To determine which kinetic classes of proteins were expressed in EHV-1-infected cells, a double immunofluorescence staining was performed on cells fixed in 100 % methanol at 220 uC for 20 min. Cells were incubated for 1 h at 37 uC with a rabbit polyclonal anti-IEP (diluted 1 : 1000) (Smith et al, 1994;Jang et al, 2001) to detect IEP expression and either mouse anti-EICP22 K2 to IR4 (EICP22) protein (1 : 500), anti-gB (clone 4B6, 1 : 100) or anti-gC (clone 1B6, 1 : 100) mAb against early (EICP22) and late gB and gC protein expression, respectively. The IEP and EICP22 antibodies were kindly provided by Dr D. J. O'Callaghan (Louisiana State University, USA).…”