Regulation of the herpesvirus saimiri (HVS) delayed-early 110-kilodalton promoter by HVS immediate-early gene products and a homolog of the Epstein-Barr virus R trans activator

Abstract

We have reported previously the detection of two stable immediate-early (IE) transcripts that accumulate in cycloheximide-treated cells infected with herpesvirus saimiri (HVS). These are the 1.6-kb mRNA from the 52-kDa gene (which is homologous to the BSLF2-BMLF1 gene of Epstein-Barr virus) and the 1.3-kb mRNA from the HindM-G fragment of virus DNA. In order to study the roles of the HVS IE gene products in the progression of a lytic infection, the promoter region of the delayed-early 110-kDa gene of HVS was sequenced, the transcription initiation site was mapped by RNase protection, and the promoter sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. Sequences between -447 and +37 (relative to the 110-kDa transcription initiation site) were sufficient for response to HVS superinfection of transfected cells, but the 110-kDa promoter was activated only poorly by the 52-kDa and HindIll-G IE (IE-G) proteins in cotransfection experiments. However, a distinct region of the genome, EcoRI-D (15 kbp), was able to activate 110-kDa-CAT expression relatively efficiently in similar experiments. A 4.7-kbp PstI fragment encoding this function was isolated and sequenced, and further subcloning identified the gene encoding the EcoRI-D trans activator. This gene, which we now designate HVS.R, is homologous to the BRLFl-encoded transcriptional effector of Epstein-Barr virus.

The herpesvirus saimiri ORF50 gene, encoding a transcriptional activator homologous to the Epstein-Barr virus R protein, is transcribed from two distinct promoters of different temporal phases

“…The EcoRI-D or HVS R protein transactivator (ORF50) is homologous to the BRLF1 gene product (also known as R or Rta) (23). BRLF1 is translated from a bicistronic mRNA which also encodes another IE gene product, BZLF1 (12,20).…”

“…These two EBV gene products function in activating expression from a number of viral (3,5,8,13,15,18,28) and perhaps cellular (7) promoters, thereby initiating the lytic cascade. However, HVS R RNA is expressed as an early transcript in HVS-infected cells (23,25). The HVS R gene is proposed to be spliced with more than one exon, although the initiation codon has not been mapped.…”

“…The HVS R gene is proposed to be spliced with more than one exon, although the initiation codon has not been mapped. Despite the likelihood of a spliced transcript, there is evidence that a DNA sequence within the identified exon is able to produce a functionally active protein capable of activating a DE ORF6 gene promoter, a component of the major DNA binding protein (23), which suggests that there may be more than one transcript produced. In this report we characterize the intron-exon structure of ORF50 and demonstrate that two transcripts are produced from two separate promoters which are active at different stages of the virus replication cycle.…”

“…Previously published transient-transfection experiments have demonstrated that a HincII fragment within the second exon of ORF50 is sufficient to transactivate the DE ORF6 promoter (23). We considered the possibility that, in addition to the promoter identified by 5Ј RACE, ORF50 contains a second transcriptional start site and promoter within the HincII fragment.…”

Primary structure of the herpesvirus saimiri genome

“…A number of other genes of herpesvirus saimiri whose primary structures were determined are not conserved in the herpesvirus family; these include U-RNA genes (4,13,65,78,110), a dihydrofolate reductase (DHFR) gene (108), a thymidylate synthase (TS) gene (14, 45), a gene for putative complement-regulating viral glycoproteins (3), a gene for an immediate-early protein with sequence similarity to the minor lymphocyte-stimulatory (Mls) proteins of mice (85,107), and two reading frames encoding homologs of cyclins and G protein-coupled receptors (82). Earlier sequencing studies showed that the genomes of herpesvirus saimiri and EBV are predominantly collinear (2, 16,38,39,46,81,83,84) and that homologous genes are in approximately equivalent locations and the same relative orientations. In this article, we present the complete DNA sequence of the coding L-DNA of herpesvirus saimiri and discuss the structural and biological relevance of the majority of the genes.…”