Characterization of the regulatory functions of varicella-zoster virus open reading frame 4 gene product

Abstract

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a protein with a predicted molecular weight of 51,540 presenting amino acid sequence homology with the immediate-early regulatory protein ICP27 of herpes simplex virus type 1. To investigate the regulatory properties of the ORF4 gene product, we performed a series of transient expression assays in Vero cells, using a plasmid expressing ORF4 as effector and several VZV genes and heterologous genes as targets. The VZV target plasmids contained promoter/regulatory regions from genes belonging to the three putative VZV kinetic classes fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target plasmids consisted of promoter/regulatory regions of human cytomegalovirus, Rous sarcoma virus, and human immunodeficiency virus type 1 fused to the reporter gene. These experiments demonstrated that the ORF4 gene product activated expression of ORF62 in a dose-dependent fashion but had no effect on the expression of the three other putative immediate-early genes (ORF4, ORF61, and ORF63). When various amounts of ORF4 were transfected in the presence of early gene promoters, dose-dependent transactivation was evidenced with the thymidine kinase gene (ORF36) and the major DNA-binding protein gene (ORF29) promoters; interestingly, little activity was detected with the promoter of the DNA polymerase gene (ORF28). No activation of late gene expression, represented by the glycoprotein I and glycoprotein II genes, was seen even over a wide range of concentrations of input ORF4 plasmid. Expression of pCMVCAT, pRSVCAT, and pHWVCAT was also stimulated by the ORF4 gene

Oka varicella vaccine is distinguishable from its parental virus in DNA sequence of open reading frame 62 and its transactivation activity

“…It has been reported that the amount of gC expressed in HEL cells infected with Oka vaccine virus is less than in cells infected with wild-type virus, and the same trend has been observed for mRNA levels [Kinchington et al, 1990;Ling et al, 1991], suggesting that the expression level of gC influences viral pathogenicity [Moffat et al, 1998]. No nucleotide changes, however, were demonstrated in the DNA sequence of gene 14, that is one of the putative late genes encoding the gC protein, in the Oka vaccine compared with its parental virus (data not shown), and there were also no nucleotide changes in genes 4, 10, 61, and 63, all of whose products are known to transregulate VZV genes [Perera et al, 1992;Defechereux et al, 1993;Schoonbroodt et al, 1996]. Next, nucleotide sequences were determined for gene 62 of the Oka vaccine and its parental virus by sequencing a region from 125 bases up- stream of the transcription start site (TTTTAA) to 23 bases downstream of the polyadenylation signal site (AATAAAA).…”

Enhancement of varicella-zoster virus infection in cell lines expressing ORF4- or ORF62-encoded proteins

“…Transactivating properties of the stably expressed proteins were monitored in transient expression assays where ORF4-expressing cells (named Vero-IE4) were transfected with 2 pg of pTKCAT target plasmid, containing the VZV thymidine kinase (TK) gene promoter and where ORF62-expressing cells (named Vero-IE62) were transfected with 2 pg of p61CAT target plasmid, containing the VZV ORF61 gene promoter. Previous studies in Vero cells have shown that these promoters are transactivated by transiently expressed ORF4p and ORF 62p [Defechereux et al, 1993;Baudoux et al, 19951.…”

“…The VZV ORF4 encodes a putative IE protein, an HSV-1 ICP27 homolog, which seems to act only as an activator of various promoters. ORF4p activates gene expression driven from VZV or other viral promoters [Defechereux et al, 1993;Nagpal and Ostrove, 19911, especially the ORF62 promoter, and has little or no effect on the expression of the other three putative VZV IE genes (ORF4, ORF61, ORF63). It also transactivates early gene promoters in a dose-dependent fashion, especially the thymidine kinase gene (ORF36) and the major DNA-binding protein gene (ORF29) promoters, while no activation of late gene promoters was observed [Defechereux et al, 19931. The ORF62p, identified as the major regulatory protein [Disney and Everett, 1990;Disney et al, 1990;Perera et al, 19921, has been shown to be the major component of the virion tegument [Kinchington et al, 19921 and is able to transactivate both immediate-early and early gene promoters in transient expression assays pRcORF4 and pRcORF62 respectively.…”

“…It also transactivates early gene promoters in a dose-dependent fashion, especially the thymidine kinase gene (ORF36) and the major DNA-binding protein gene (ORF29) promoters, while no activation of late gene promoters was observed [Defechereux et al, 19931. The ORF62p, identified as the major regulatory protein [Disney and Everett, 1990;Disney et al, 1990;Perera et al, 19921, has been shown to be the major component of the virion tegument [Kinchington et al, 19921 and is able to transactivate both immediate-early and early gene promoters in transient expression assays pRcORF4 and pRcORF62 respectively. Construction of p62CAT, pTKCAT, and p61CAT has been described elsewhere [Defechereux et al, 1993;Disney et al, 1990;Mc Kee et al, 19901. Inschauspe et al, 1989;Perera et al, 19921.…”

Varicella-zoster virus open reading frame 4 encodes an immediate-early protein with posttranscriptional regulatory properties

“…The ORF4 gene product (51 kDa) is a transactivator of gene expression (10,21,35,39,40) which presents sequence similarity, especially in the carboxyl-terminal region, with ICP27 of HSV-1, an important multifunctional viral regulatory protein (30,42,44,46). It has been shown that ICP27 and the ORF4 gene product are not functionally homologous (34).…”