Mutational analysis of varicella-zoster virus major immediate-early protein IE62

Abstract

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3.

Intracellular distribution of the ORF4 gene product of varicella-zoster virus is influenced by the IE62 protein

“…IE62 was expressed from pSV62 (Disney et aI., 1990). pHN570, pHNII37 and p,X612-865, which are insertion and deletion mutants of IE62, are described in Baudoux et al (1995); mutant pHN570 contains an in-flame insertion after codon 570 of IE62 and mutant pHN1137 contains an in-frame insertion after codon 1137 of IE62. The in-frame deletion mutant pA612-865 was constructed by removing the region extending from codon 612 to 865 in the IE62 protein using HpaI and NruI restriction enzymes.…”

“…These differences in staining might be partially explained by the transfection conditions and do not necessarily reflect normal infection patterns because of the absence of interactions and regulation by other virus proteins. Cells individually transfected with pSV62 showed essentially diffuse nuclear fluorescent staining (Baudoux et al, 1995).…”

“…Cooperative interactions amongst VZV putative immediate-early proteins are likely events (Perera et aI., 1992a;Hay & Ruyechan, 1994). IE62, defined as a major regulatory protein, can transactivate the expression of VZV genes of all three putative kinetic classes (immediate-early, early and late) Perera et al, 1992a, b;Baudoux et al, 1995) and autoregulate its promoter Perera et al, 1992b). IE4, which possesses transactivating properties on various promoters when transfected alone, can enhance activation by IE62 when both proteins are expressed Perera et al, 1992a;Defechereux et al, 1993;Moriuchi et al, 1994).…”

“…VZV IE62 is located in nuclei of infected (Forghani et al, 1990) and transfected cells (Baudoux et al, 1995), whereas IE4 is mainly distributed in the cytoplasm (Moriuchi et al, 1995). A possible means for IE4 to perform some of its regulatory functions is to be transported into nuclei by other proteins such as IE62.…”

Combinatorial Transcription of Herpes Simplex Virus and Varicella Zoster Virus Immediate Early Genes Is Strictly Determined by the Cellular Coactivator HCF-1

“…4A). VZV differs from HSV, however, in that the IE62 protein also functions as an autostimulatory transcription factor (36,38) that is copackaged in the VZV virion (39). Therefore, to determine the potential role of HCF-1 in the ORF10 and IE62 regulation of the VZV IE gene, reporter constructs containing the VZV EC element and the VZV IE62 responsive promoter were transfected into control and HCF-1-depleted cells in the presence or absence of ORF10 or IE62.…”