Interaction between the Varicella Zoster Virus IE62 Major Transactivator and Cellular Transcription Factor Sp1
Journal of Biological Chemistry
The varicella zoster virus (VZV) IE62 protein is involved in the activation of expression of all three kinetic classes of VZV proteins. Analysis of the viral promoter for VZV glycoprotein I has shown that the cellular factor Sp1 is involved in or required for the observed IE62 mediated activation. Co-immunoprecipitation experiments show that the two proteins are present in a complex in VZV-infected cells. Protein affinity pull-down assays using recombinant proteins showed that IE62 and Sp1
... t IE62 and Sp1 interact in the absence of any other viral and cellular proteins. Mapping studies using GST-fusion proteins containing truncations of IE62 and Sp1 have delimited the interacting regions to amino acids 612-778 in Sp1 and amino acids 226 -299 in IE62. The region identified in Sp1 is involved in DNA-binding, synergistic Sp1 activation, and Sp1 interaction with cellular transcription factors. The interacting region identified in IE62 overlaps with or borders on sites involved in interactions with the VZV IE4 protein and the cellular factors TBP and TFIIB. Assays using wild-type and mutant promoter elements indicate that Sp1 is involved in recruitment of IE62 to the gI promoter and IE62 enhances Sp1 and TBP binding. Varicella zoster virus (VZV) 1 is a member of the alphaherpesvirinae and the causative agent of chicken pox (varicella) and shingles (zoster). The VZV genome is a linear doublestranded DNA molecule, which encodes approximately seventy proteins (1). The entire complement of VZV genes is believed to be expressed during lytic infection in three broad kinetic classes, immediate early (IE), early (E), and late (L). Transcription of VZV genes is performed by the host cell RNA polymerase II, as is the case with all other herpes viruses. Efficient expression of the VZV genome is driven by a small group of VZV gene products including those encoded by open reading frames (ORFs) 62, 4, 61, 63, and 10 (2-11). The major viral transacti-vator is the product of ORF 62 and its complement, ORF 71, which lie within the inverted repeats bracketing the Us region of VZV DNA. This protein is commonly designated IE62 since it is synthesized in the immediate early phase of lytic VZV gene expression. IE62 contains a potent N-terminal acidic transactivation domain and is capable of activating the expression of all three kinetic classes of VZV genes (12-14) . While IE62 is involved in transactivation of VZV promoters, careful analysis of a limited number of individual viral promoters has shown that cellular transcription factors acting at sites upstream of the coding regions of the viral genes are also involved in the mechanism of IE62 activation. These proteins include the ubiquitous, sequence specific cellular factor Sp1. Sp1 is the protoype of a family of closely related factors which bind to GC-rich elements including the GC-box (GGGCGG or GGGCGGG) and the related GT/CACCC-box. Sp1 contains five distinct domains, four of which (A, B, C, and D) are involved in various aspects of transcriptional activation as well as a DNA binding region containing three zinc fingers (reviewed in . Sp1 interacts with the TATA-binding protein (TPB) and the TBP-associated factor TAF 110/130 via the glutamine rich A and B domains (18, 19). Sp1 in addition interacts physically and/or functionally with a variety of other cellular proteins involved in transcription including p53, p300, cJun, RelA, the p107 Rb-related protein, smads 3 and 4, the promyelocytic leukemia protein (PML), the retinoic acid receptor (RAR/RXR), COUP-TF, the von Hippel-Lindau tumor suppressor gene product, YY1 and E2F (20 -34). Sp1 has also been shown to interact functionally with several viral transcriptional regulatory proteins. These include the E2 protein from bovine and human papilloma viruses (with which a direct physical interaction has been demonstrated), the HIV-1 Vpr protein, the Tax protein of human T cell leukemia virus type 1 (HTLV-1), the parvovirus NS-1 protein, and the Herpes Simplex Virus type 1 (HSV-1) transactivator, . Recent studies from this and other laboratories have shown that Sp1 binding sites are important for the regulation of expression of VZV promoters (42, 43). In the work described here we have explored the involvement of Sp1 in IE62-mediated activation of the viral glycoprotein I (gI) promoter using reporter gene assays and site-specific mutations and show that the presence of an Sp1 site is important for this activation. The presence of an Sp1-IE62 complex in infected cell extracts has been demonstrated by co-immunoprecipitation and the regions of these two proteins, which interact have been mapped by protein affinity pull-down assays. Finally, we present data, which indicate that Sp1 is involved in recruiting IE62 to the gI promoter and that the presence of IE62 increases the levels of Sp1 and TBP binding to the promoter.