Identification and transcriptional analyses of the UL3 and UL4 genes of equine herpesvirus 1, homologs of the ICP27 and glycoprotein K genes of herpes simplex virus

Abstract

The DNA sequence of 3,240 nucleotides of the XbaI G fragment located in the unique long (UL) region of the equine herpesvirus 1 genome revealed two major open reading frames (ORFs) designated UL3 and UL4. The UL3 ORF of 470 amino acids (aa) maps at nucleotides (nt) 4450 to 3038 from the long terminus, and its predicted 51.4-kDa protein product exhibits significant homology to the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1; 32% identity) and to the ORF4 protein of varicella-zoster virus (13% identity). Interestingly, a zinc finger motif is conserved in the C-terminal domains of both ICP27 of HSV-1 (aa 483 to 508) and UL3 of equine herpesvirus 1 (aa 441 to 466). The UL4 ORF of 343 aa maps at nt 5618 to 4587 and could encode a protein of 38.1 kDa which exhibits significant homology to the UL53 protein (cell fusion protein or glycoprotein K) of HSV-1 (26% identity) and to the ORF5 protein of varicella-zoster virus (33% identity). Analyses of the UL4 amino acid sequence revealed domains characteristic of a membrane-bound glycoprotein and included potential signature sequences for (i) a signal sequence, (ii) two N-linked glycosylation sites, and (iii) four transmembrane domains. Nucleotide sequence analyses also revealed potential TATA boxes located upstream of the UL3 and UL4 ORFs. However, only a single polyadenylation signal (nt 2988 to 2983) was detected downstream of the UL3 ORF. Northern (RNA) blot hybridization and Si nuclease analyses were used to map and characterize the UL3 and UL4 mRNAs. Metabolic inhibitors were used to identify the kinetic class of these two genes. The data revealed that UL3 is an early gene that encodes a 1.6-kb mRNA, while UL4 is a late gene encoding a 3.8-kb mRNA that overlaps the UL3 transcript. Both transcripts were shown by Si nuclease analyses to initiate 24 to 26 nt downstream of their respective TATA boxes and to have a common transcription termination signal as a pair of 3'-coterminal mRNAs.

Expression of an equine herpesvirus 1 ICP22/ICP27 hybrid protein encoded by defective interfering particles associated with persistent infection

“…The IR4 protein is ex- pressed abundantly and localizes predominantly to the nucleus (26,27), and transient expression assays demonstrate that the IR4 protein can activate a variety of EHV-1 early and late promoters in the presence of other EHV-1 trans-activators, such as the immediate-early protein and the UL3 protein (28). The UL3 protein exhibits significant homology to the ICP27 immediate-early regulatory protein of HSV-1 (32% identity) and to the ORF4 protein of varicella-zoster virus (13% identity) (66). Studies with ICP27 temperature-sensitive and deletion mutants have demonstrated that HSV-1 ICP27 possesses the ability to down-regulate the immediate-early gene and some early genes and to up-regulate late gene expression (44-46, 56, 57, 60).…”

“…The 8-nucleotide (nt) recombination site was confirmed on a pDI2227 subclone. This ORF (designated as IR4/UL3 for the hybrid of IR4 and UL3) appears to have the transcription initiation site of the IR4 gene, which gives rise to an early mRNA and a late mRNA, as described by Holden et al (27), and the polyadenylation signal of the UL3 gene, as described by Zhao et al (66). A comparison of the IR4 sequences in the hybrid ORF with those in the IR4 gene revealed a 28-bp deletion in the upstream noncoding region, in which 4 of the 17 seven-mer repeats were missing in pDI2227.…”

“…S1 nuclease protection assay. The protocol for the S1 nuclease protection assay used in this laboratory has been described elsewhere (22,27,66). Molecular weight markers included a radiolabeled fragment mixture from cleavage of pUCBM21 DNA with the restriction endonucleases HpaI and DraI-HindIII, which were purchased from Boehringer Mannheim Biochemicals, Indianapolis, Ind.…”

Pseudorabies Virus Glycoprotein K Requires the UL20 Gene Product for Processing

“…Mature glycoprotein K (gK) of PrV is a hydrophobic protein of 36 kDa which is present in virions and contains both highmannose as well as complex forms of N-linked glycans (25). The protein is conserved among the alphaherpesviruses, and homologous proteins or open reading frames (ORFs) have also been described for herpes simplex virus types 1 and 2 (HSV-1 and -2), bovine herpesvirus 1, equine herpesvirus 1, varicella-zoster virus (VZV), infectious laryngotracheitis virus, and Marek's disease virus (12,17,20,32,34,35,39). All deduced gK homologous proteins are characterized by the presence of four hydrophobic domains of sufficient length to be membrane spanning.…”

The ICP22 protein of equine herpesvirus 1 cooperates with the IE protein to regulate viral gene expression

“…During a productive lytic infection, the genes of EHV-1 are coordinately expressed and temporally regulated in immediate-early (IE), early (E), and late (L) fashions (15,16). EHV-1 EICP22 (previously named IR4; ICP22 homolog) (23), EICP27 (previously named UL3; ICP27 homolog) (63), and ICP0 (3) are regulated as E genes, in contrast to the case for herpes simplex virus type 1 (HSV-1), in which they are members of the IE gene family (2,26,27,31,40,58).…”