Search for physical interaction between BICP0 of bovine herpesvirus-1 and p53 tumor suppressor protein.

The immediate-early (IE) protein BICP0 of bovine herpesvirus-1 (BHV-1) may have other functions besides transactivation of viral promoters. Recently, we observed that BICP0, delivered to cultured cells by a helpervirus-free amplicon system, forms spherical or doughnut-like structures in which the tumor suppressor protein p53 is sequestered. The objective was to determine whether BICP0 and p53 interact physically, we used both yeast and mammalian two-hybrid systems. As a bait plasmid, pVA3 which encodes a hybrid protein consisting of the Gal4 DNA binding domain fused to murine p53 was used. The BICP0 gene or its truncated versions were inserted into the prey plasmid pGAD424. Bait and prey plasmids were cotransformed into yeast strain Y153, which has LacZ and HIS3 reporter genes under the control of Gal4 upstream activating sequence. After 4-6 days, colonies were stained for beta-galactosidase activity. In the mammalian two-hybrid system, pM-53 was used as a bait where truncated p53 fused to Gal4 DNA binding domain is expressed. The BICP0 gene was cloned into prey plasmid pVP16. The interaction between p53 and SV40 T-antigen was evaluated as a positive control in both systems. Neither full-length BICP0 nor its truncated derivatives induced beta-galactosidase activity in yeast whereas the positive control turned blue under the same conditions. The mammalian two-hybrid system, in which chloramphenicol acetyltransferase (CAT) activity was used as a reporter, also failed to show an interaction between these two proteins. Co-localization of p53 with BICP0 in spherical structures is unlikely to result from a direct physical interaction between these two proteins. Mediation by additional cellular proteins may be required.

Transduction of Vero cells and bovine monocytes with a herpes simplex virus-1 based amplicon carrying the gene for the bovine herpesvirus-1 Circ protein.

Herpes simplex virus-1 (HSV-1) based amplicon vectors are promising gene delivery vehicles because they have a large transgene capacity and can efficiently transduce many different cell types, including non-dividing cells, of various animal species. The Circ protein of bovine herpesvirus-1 (BHV-1) is a myristylated virion component of unknown function. Preliminary experiments with a circ gene deletion mutant indicated that Circ may influence the host's immune response by downregulating MHC-II expression in bovine monocytes. To get more insight into the function of Circ, amplicon vectors were constructed with various open reading frames (ORFs) under the control of the HSV-1 IE4/5 promoter: (i) the Circ ORF alone, (ii) a fusion ORF encoding an N-terminal Circ fused to the enhanced green fluorescent protein (eGFP), (iii) the eGFP ORF alone, and (iv) the Circ ORF in the inverted orientation. Upon helpervirus-free packaging into HSV-1 amplicon particles and transduction of Vero cells, both Circ alone and the Circ-eGFP fusion protein produced a punctate pattern within the cytoplasm, suggesting membrane association of the myristylated protein. In contrast, eGFP alone was evenly distributed over the cytoplasm of transduced cells. Upon infection of bovine buffy-coat cells, it was observed that cells of the monocyte lineage but not lymphocytes were transduced. Transgene expression reached a peak around 20h after transduction and lasted for at least 90h. Transduced monocytes underwent specific morphological changes, which may be attributed to Circ synthesis.

Sequence analysis of the bovine herpesvirus type 1 genes homologous to the DNA polymerase (UL30), the major DNA-binding protein (UL29) and ICP18.5 assembly protein (UL28) genes of herpes simplex virus.

The nucleotide sequence of a 10.5 kb region (map position 0.332 to 0.410) of bovine herpesvirus type 1 (BHV-1) was determined. This region contained three open reading frames (ORFs) homologous to herpes simplex virus DNA polymerase catalytic subunit (DNApol, UL30), major DNA-binding protein (MDBP, UL29) and ICP18.5 assembly protein (ICP18.5, UL28). The BHV-1 DNApol. MDBP and ICP18.5 ORFs were 1246, 1203 and 826 amino acids long with a calculated molecular mass of 134.2 kDa, 124.4 kDa and 86.9 kDa, respectively. They showed a high homology with alphaherpesvirus homologs despite large differences in the G + C content of the UL30-UL28 segment ranging from 44.4% for varicella zoster virus to 71.5% for BHV-1. Particularly well conserved among Alphaherpesvirinae are the putative functional domains of the DNApol and MDBP proteins which are discussed. Phylogenetic analysis revealed that BHV-1 clustered in the Varicellovirus genus with the animal D-type viruses. In this group, the BHV-1 position was shown to vary according to the investigated genes. Indeed, pseudorabies virus clustered with BHV-1 in the DNApol tree but with equine herpesvirus 1 in the ICP18.5 tree.

Immediate-early protein BICP22 of bovine herpesvirus 1 trans-represses viral promoters of different kinetic classes and is itself regulated by BICP0 at transcriptional and posttranscriptional levels.

Bovine herpesvirus 1 (BHV-1) encodes four immediate-early (IE) proteins. The transactivators BICP0 and BICP4 are key regulatory elements in viral replication, and circ is a myristylated virion component, whereas BICP22--originating from a spliced 1.7-kb transcript synthesized with dual IE and late kinetics--has not yet been characterized as a protein. In this study, Western blot and immunofluorescence analysis using antisera against a C-terminal oligopeptide revealed major 50-kDa and minor 35-kDa species of BICP22, predominantly located in the nuclei of BHV-1 infected cells. In transient expression assays, BICP22 acted as transrepressor protein on viral promoters of different kinetic classes, e.g. the IE promoter of the BICP4/BICP0 gene, early promoter of the BICP0 gene, and late promoter of the gC gene. The BICP22 gene promoter itself was not repressed by BICP22; it could be dissected into a proximal region stimulated by BICP0 and a distal region stimulated by BHV-1 alpha-transinducing factor. Replacement of the BICP22 promoter by cytomegalovirus IE promoter revealed an additional posttranscriptional level of regulation whereby more BICP22 accumulated in cells when functional BICP0 was present. Interplay of BICP22 and BICP0 might involve the recently described nuclear domains (ND10) and ubiquitin-dependent pathway.

Recombinant bovine herpesvirus-1 (BHV-1) lacking transactivator protein BICPO entails lack of glycoprotein C and severely reduced infectivity.

The immediate-early transactivator protein BICPO is a key regulatory element of bovine herpesvirus 1 (BHV-1) replication based on transient expression assays. To examine BICPO function in the context of the viral genome, we created recombinant BHV-1 expressing beta-galactosidase instead of BICPO. To complement the defect, a neomycin resistant MDBK cell line (M164) expressing BICPO was established, permitting selection of a blue-staining BHV-1 recombinant (A2G2). Southern blot and PCR analysis confirmed that the BICPO gene was interrupted by the beta-galactosidase gene and that wt progeny was absent. Compared with wt BHV-1, A2G2 reached lower titers in M164 cells but replicated with similar kinetics. Once isolated, A2G2 also grew in MDBK cells although the titer was reduced a further 10-fold and the virus remained strongly cell-associated. Thus, BICPO is not absolutely required for replication in cell culture. Gene expression of A2G2 was investigated by Western blots and immunofluorescence. Surprisingly, not only was BICPO absent, but glycoprotein C (gC) was also missing. Other viral genes were expressed normally. Semiquantitative PCR showed that A2G2 produced similar amounts of viral DNA as wt but a much smaller number of infectious particles. Cotransfection of A2G2 DNA and a plasmid containing the BICPO gene yielded revertant virus with fully restored wt properties. We conclude that BICPO is required for gC expression, and that the missing gC partly accounts for the reduced A2G2 infectivity.

Molecular virology of ruminant herpesviruses.

Molecular virology has served to establish bovine herpesvirus 1 (BHV-1) as the prototype member of ruminant herpesviruses. Based on the genomic sequence of the virus, we aim to identify and characterize virus-specified components, to explain their concerted action, and to predict how the chain of events during the lytic and latent phases of the viral life cycle may be interrupted. The nucleotide sequence of the BHV-1 genome (136 kb) has just been completed by international cooperation (July 1995; except for a small gap in UL36). It comprises 67 unique genes and 2 genes, both duplicated, in the inverted repeats. In general, these genes exhibit strong homology at the amino acid sequence level to those of other alphaherpesviruses (HSV-1, VZV, EHV-1) and are arranged in similar order. A few genes are peculiar to only one or two herpesviruses, e.g. in BHV-1 the circ, UL0.5, UL3.5 and US1.5 genes. Not long ago, the repertoire of BHV-1 proteins under study was restricted to the three major glycoproteins (gB, gC, and gD) and thymidine kinase. The repertoire is now growing rapidly and includes 7 additional glycoproteins (gE, gI, gH, gL, gG, gK and gM), a number of enzymes (e.g. ribonucleotide reductase, DNA Polymerase, dUTPase), and a group of regulatory proteins (BICPO, 4, 22, and 27, alpha TIF). Investigations into the functions of these proteins and comparison with their counterparts in other herpesviruses should reveal which are useful targets for diagnosis, prevention or antiviral treatment. Recombinant viruses containing deletions or replacements of individual genes are being created, aiming at vaccine development and insights into pathogenesis, notably latency, neurotropism, and interference with host functions. Molecular analysis of other ruminant herpesviruses is much less advanced. Over a dozen virus species have been described; most share basic properties with BHV-1 and may be classified as alphaherpesviruses. The gammaherpesviruses are represented by the proposed agent of malignant catarrhal fever, alcelaphine herpesvirus 1, and by bovine herpesvirus 4, whose partial sequences exhibit similarity to herpesvirus saimiri.

Gene contents in a 31-kb segment at the left genome end of bovine herpesvirus-1.

We report the nucleotide sequence of a 31-kb segment at the left genome end of bovine herpesvirus-1 (BHV-1) and show that it comprises 19 different open reading frames (ORFs), including seven which have been described previously (circ, dUTPase, UL49.5, alpha TIF, VP8, glycoprotein C, and ribonucleotide reductase small subunit). The new sequence resulted in a correction at the C-terminus of glycoprotein C. All 19 ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 (HSV-1) in the range of the UL54 to UL37 genes. No BHV-1 homologs of the HSV-1 UL56, UL55, and UL45 genes were identified. The BHV-1 circ gene was the only gene without a HSV-1 counterpart. The additional ORFs 1 and 2 found at the left genome end of equine herpesvirus-1 (EHV-1) were absent in BHV-1. Among the newly sequenced BHV-1 ORFs are homologs of ICP27 (UL54), glycoprotein K (UL53), helicase-primase (UL52), DNA polymerase accessory protein (UL42), ribonucleotide reductase large subunit (UL39), and several virion proteins (UL49, UL46, UL43, UL41, UL38, UL37), most of which are strongly conserved in all herpesviruses. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed.

Identification and characterization of BICP27, an early protein of bovine herpesvirus 1 which may stimulate mRNA 3' processing.

Sequence analysis of the left genomic terminus of bovine herpesvirus 1 (BHV-1) revealed two convergently transcribed genes with 3' ends about 300 bp apart. The gene on the left is the previously described circ gene; that on the right was found to encode a protein of 400 amino acids which was designated BICP27 because of its homology to ICP27 (Vmw63) of herpes simplex virus 1 (HSV-1) and related proteins from other alpha- beta- and gammaherpesviruses. Rabbit antisera raised against a synthetic oligopeptide representing the amino terminus of the predicted polypeptide demonstrated the presence of BICP27 in the nuclei of infected cells by in situ immunoadsorbent assays. In Western immunoblots, BICP27 was detected as a 50 kDa BHV-1 specific protein expressed with early kinetics, in contrast to HSV-1 ICP27 which is an immediate early (IE) protein. A DNA fragment containing BICP27 coding sequences was inserted into a baculovirus genome. The recombinant BICP27 protein, identified by its reactivity with the antipeptide sera, exhibited the same electrophoretic mobility as BICP27 specified by BHV-1. Transient expression assays using target genes differing only in their poly(A) sites showed that BICP27, like its HSV-1 counterpart, may be involved in 3' processing of mRNA.

Diagnosis of fetal infection with porcine parvovirus by in situ hybridization.

In situ hybridization (ISH) for the diagnosis of fetal infection with porcine parvovirus (PPV) was compared with immune electron microscopy (IEM) and serology by immunofluorescence (IF) for its sensitivity and its applicability in a routine diagnostic laboratory. The technique was applied to the examination of sections of formalin-fixed paraffin-embedded tissues from 68 fetuses. Fifty-three of these fetuses were diagnosed serologically since they had a crown rump length of more than 17 cm, i.e. they were mature enough to mount a humoral immune response; 38 were positive and 15 negative. Eleven out of 15 smaller fetuses examined for the presence of viral antigen by immune electron microscopy (IEM) were positive and 4 were negative. Heart and lung were found to be the most suitable organs for in situ hybridization. In situ hybridization yielded a positive result in 8 of the 11 IEM positive fetuses and in 33 of the 38 serologically positive fetuses. No signal was detected in any of the 4 IEM or the 13 serologically negative fetuses. Expenses for IEM were estimated to be 179% of the expenses for ISH. Expenses for serology by IF on the other hand were 67% of the expenses for ISH. From this it was concluded that the most efficient way to diagnose a fetal infection with PPV was serology by IF, if possible with samples from several fetuses and that the other techniques, IEM or ISH, ought to be reserved for those cases where no immunocompetent fetuses were available for diagnosis.

Sequence analysis of the UL39, UL38, and UL37 homologues of bovine herpesvirus 1 and expression studies of UL40 and UL39, the subunits of ribonucleotide reductase.

We sequenced the region of the bovine herpesvirus type 1 (BHV-1) genome corresponding to map units 0.172-0.230 (7964 bp), representing the UL39, UL38, and UL37 homologues of herpes simplex virus which encode the large subunit of ribonucleotide reductase (RR) and components of the viral capsid and the tegument, respectively. To discriminate between two potential initiator AUGs of the UL39 gene, the 5' end of the mRNA was mapped by S1 nuclease protection assays. Comparison of the amino acid sequences of the three BHV-1 proteins with analogous polypeptides from several other herpesviruses revealed significant levels of homology. We also compared the expression kinetics of the large (R1, UL39) versus the small (R2, UL40) RR subunits during the course of in vitro BHV-1 infection by Western blotting using specifically developed and calibrated antisera. Our results show that the R1 protein was synthesized earlier than its R2 counterpart. Moreover, the R1 protein accumulated to a higher level than the R2 protein even though the R2 transcript was in greater abundance than the R1 mRNA. This is discussed with regard to the translational efficiency of their transcripts.

Nucleotide sequence analysis of a 30-kb region of the bovine herpesvirus 1 genome which exhibits a colinear gene arrangement with the UL21 to UL4 genes of herpes simplex virus.

We report the nucleotide sequence of the 19-kb HindIII fragment B of bovine herpesvirus 1 (BHV-1) DNA and adjacent parts of the HindIII A and L fragments, which together span a still completely uncharted 30-kb region located between the glycoprotein H gene and the right end of the unique long segment. The analysis revealed 17 complete open reading frames (ORFs) and 2 ORFs that were interrupted by potential splice donor and acceptor sites. All of these ORFs exhibited strong amino acid sequence homology to the gene products of other alphaherpesviruses. The BHV-1 ORFs were arranged colinearly with the prototype sequence of herpes simplex virus 1 in the range of the UL21 to UL4 genes. Colinearity was also observed with the genes of betaherpesviruses and gamma herpesviruses, although not all ORFs exhibited clear sequence homology. The possible functions of the proteins encoded within the sequenced region are assessed and features found are discussed. Unexpected findings include the following: high amino acid sequence conservation among alphaherpesviruses despite large differences in G + C content, ranging from 45% for varicella zoster virus to 72% for BHV-1; high similarity with other UL20 proteins at the predicted structural level in spite of relatively low amino acid homology; and a 2-kb open reading frame overlapping UL19 in the opposite sense and exhibiting high amino acid similarity to the same area of pseudorabies virus.

Identification of the bovine herpesvirus 1 circ protein, a myristylated and virion-associated polypeptide which is not essential for virus replication in cell culture.

We have recently reported immediate-early (IE) transcription over covalently joined genome ends of bovine herpesvirus 1 (BHV-1). A spliced 1.5-kb IE RNA (IER1.5) is coterminal with an unspliced 1.1-kb late RNA (LR1.1) which is transcribed from the left end of the genome. Sequence analysis reveals an open reading frame common to IER1.5 and LR1.1 predicted to encode the 247-amino-acid circ polypeptide. This paper reports on the identification of circ as a protein. Using a rabbit antiserum raised against a synthetic oligopeptide representing the carboxy terminus of the predicted circ polypeptide for Western blot (immunoblot) analyses and immunofluorescence assays, we identified a 34-kDa virion-associated protein which accumulated in the cytoplasm of infected cells. To confirm that LR1.1 indeed encoded the 34-kDa polypeptide, we inserted a DNA fragment containing circ coding sequences into the Autographa californica baculovirus genome. A group of recombinant polypeptides with sizes of 32, 34, and 35 kDa were identified by their reactivity with the antipeptide serum. Chicken egg yolk antibodies raised against total proteins of insect cells infected with the recombinant baculovirus identified the 34-kDa circ protein specified by BHV-1. The recombinant circ polypeptides and the circ protein specified by BHV-1 were both myristylated, as determined by radiolabeling with [3H]myristic acid. It was noted that the circ gene could be deleted from the BHV-1 genome without impairing virus replication in cell culture.

BICP22 of bovine herpesvirus 1 is encoded by a spliced 1.7 kb RNA which exhibits immediate early and late transcription kinetics.

Kinetic analysis of the two divergent immediate early (IE) transcription units of bovine herpesvirus 1 (BHV-1) revealed an unexpected behaviour. The IE1.7 promoter was not turned off at the end of the IE period but acted as a late promoter, unlike the adjacent IE4.2/2.9 promoter which was active only under IE conditions. The genome region specifying the IE1.7 gene was sequenced (0.814 to 0.839 map units). The IE1.7 promoter was found to overlap with duplicated sequence elements bearing close similarity to herpesvirus origins of replication, which may explain the biphasic transcription kinetics. Exons 1 and 2 of the spliced IE1.7 transcript were non-coding. Exon 3 was found to contain a single open reading frame encoding a protein of 300 amino acids that was designated BICP22 because of its homology to ICP22 (Vmw68) of herpes simplex virus type 1 and related proteins from other herpesviruses. The protein probably represents IEP-55, the most abundant BHV-1 phosphoprotein observed under IE conditions.

Identification and zinc dependence of the bovine herpesvirus 1 transactivator protein BICP0.

Bovine herpesvirus 1 (BHV-1) specifies and unspliced early 2.6-kb RNA (ER2.6) which is 3' coterminal with exon 2 of the 2.9-kb immediate-early (IE) RNA. The two transcripts have a common open reading frame (676 codons). The predicted protein, designated BHV-1 infected cell protein 0 (BICP0), contains a zinc finger domain with homology to ICP0 of herpes simplex virus type 1 and protein 61 of varicella-zoster virus, and depending on the promoter, it acts as a strong activator or as a repressor in transient expression assays. In situ immunoadsorbent assays using antisera against synthetic oligopeptides demonstrated that BICP0 accumulates in nuclei of BHV-1-infected cells, as expected for an IE gene product involved in gene regulation. Western blots (immunoblots) revealed a BHV-1-specific 97-kDa protein which was detectable during the IE phase and also at later periods of infection, indicating that the kinetics of BICP0 synthesis is consistent with the switch from IER2.9 to ER2.6. To confirm that ER2.6 encoded the 97-kDa BICP0 protein, a DNA fragment containing BICP0-coding sequences was inserted into the Autographa californica baculovirus genome. A recombinant protein, identified by its reactivity with antipeptide sera, exhibited the same electrophoretic mobility as BICP0 specified by BHV-1. We microinjected Xenopus oocytes with a BICP0 effector plasmid and a promoter-chloramphenicol acetyltransferase plasmid. BICP0-induced stimulation of this promoter was strongly reduced when intracellular zinc was chelated by thionein, indicating that the effect of BICP0 is zinc dependent.

Promoter, spliced leader, and coding sequence for BICP4, the largest of the immediate-early proteins of bovine herpesvirus 1.

We report the complete nucleotide sequence of the bovine herpesvirus 1 (BHV-1) immediate-early gene encoding BICP4, the homolog of the ICP4 protein of herpes simplex virus. Combined with previous mapping studies, the sequence analysis revealed that the transcript for BICP4 consisted of a noncoding leader RNA (exon 1; 0.35 kb) separated by an intron (0.46 kb) from the main body (exon 2; 4.1 kb). The open reading frame for BICP4 (1343 amino acid residues) started 27 nt after the splice site and extended across exon 2 for most of its length, BICP4 contained two domains of high homology (regions 2 and 4), which had been recognized earlier to be most conserved in the ICP4 homologs of alpha-herpesviruses and to be functionally important. These domains were flanked by three regions of lower but still discernible homology. Unique features of BICP4 were two large clusters of glutamic acid residues near the end of region 3, and the displacement of a polyserine tract to region 5, which in all other ICP4 homologs residues near the end of region 1. Transient expression assays showed that BICP4 repressed its own promoter and activated other herpes-virus genes. The 8.1-kb sequence summarized here completes analysis of the inverted repeats of the BHV-1 genome; it includes a segment (2.5 kb) upstream of the BICP4 gene apparently devoid of coding sequences but containing numerous scattered transcription signals.

Immediate-early transcription over covalently joined genome ends of bovine herpesvirus 1: the circ gene.

Herpesvirus genomes are linear molecules in virions. Prior to replication in host cells, they form circular templates by unknown mechanisms. Examining lytic infection with bovine herpesvirus 1, we observed immediate-early transcription over joined genome ends, which suggested that circles are present at the initial stage of infection. Among the transcripts was a spliced immediate-early RNA (1.5 kb) sharing exon 1 with previously described major immediate-early transcripts from the right genome end and exon 2 with a late transcript located near the left genome end. Exon 2 encodes a putative circ-encoded protein with homology to the varicella-zoster virus open reading frame 2 and equine herpesvirus 1 open reading frame 3 products. The novel features reported here for bovine herpesvirus 1 may constitute a more general property of herpesviruses.

Immediate-early RNA 2.9 and early RNA 2.6 of bovine herpesvirus 1 are 3' coterminal and encode a putative zinc finger transactivator protein.

Bovine herpesvirus 1 (BHV-1) contains three major immediate-early (IE) genes involved in regulation of the productive cycle of replication. Two spliced IE RNAs, IER4.2 (4.2 kb) and IER2.9 (2.9 kb), are under the control of a single promoter; IER1.7 (1.7 kb) is transcribed from a different promoter in the opposite direction. Examining the kinetics of transcription, we found that the IER4.2/2.9 promoter was turned off at the end of the IE period. An alternative promoter became active, directing synthesis of an unspliced early RNA, ER2.6 (2.6 kb), which was colinear with the second exon of IER2.9 except for its 5' end in the intron about 10 bases upstream of the splice site. Sequence analysis revealed a single open reading frame common to IER2.9 and ER2.6 with a coding potential of 676 amino acids. The putative protein, named p135, contained a cysteine-rich zinc finger domain near the N terminus with homology to ICP0 of herpes simplex virus type 1, to protein 61 of varicella-zoster virus, to early protein 0 of pseudorabies virus, and to other viral and cellular proteins. The remaining parts of p135 exhibited only limited homology, mainly with pseudorabies virus protein 0, but the entire sequence was highly conserved between two strains of BHV-1 (K22 and Jura). The latency-related antisense transcript covered a large portion of ER2.6 excluding the zinc finger coding region. In transient expression assays, p135 activated a variety of promoters, including that for ER2.6, but repressed the IER1.7 promoter. Thus, p135 combines functional characteristics of ICP0, a strong transactivator, and of protein 61, a repressor. BHV-1 seems to have evolved a subtle mechanism to ensure the continued synthesis of p135 while turning off IER4.2, which encodes p180, the herpes simplex virus type 1 ICP4 homolog.

The three major immediate-early transcripts of bovine herpesvirus 1 arise from two divergent and spliced transcription units.

Among 54 transcripts expressed in a temporal cascade during lytic infection with bovine herpesvirus 1, we have previously identified three major immediate-early (IE) RNAs, IER4.2 (4.2 kb), IER2.9 (2.9 kb), and IER1.7 (1.6 to 1.8 kb depending on the virus strain) transcribed from the HindIII C genome region (U. V. Wirth, K. Gunkel, M. Engels, and M. Schwyzer, J. Virol. 63:4882-4889, 1989). Northern (RNA) blot, S1 nuclease protection, and primer extension analysis used in the present study demonstrated that all three IE transcripts were spliced and originated from two divergent transcription units with start sites located in the inverted repeat. Transcription unit 1 encoded two alternative spliced transcripts, IER4.2 and IER2.9, with a common exon 1 located at 0.797 to 0.795 map units (m.u.) and an exon 2 for IER4.2 (0.792 to 0.762 m.u.) in the inverted repeat; exon 2 for IER2.9 (0.754 to 0.738 m.u.) was located in the unique long sequence and transcribed in antisense orientation to latency-related RNA. Transcription unit 2 (0.818 to 0.836 m.u.), further characterized by cDNA cloning, encoded the spliced IER1.7 with three exons in the inverted repeat. Additional minor IE transcripts were interpreted as unspliced precursors and splicing variants. With regard to the number and layout of IE genes, bovine herpesvirus 1 occupies an intermediate position between pseudorabies virus and equine herpesvirus 1 on the one hand and varicella-zoster virus and herpes simplex virus type 1 on the other.

Pseudorabies virus immediate-early gene overlaps with an oppositely oriented open reading frame: characterization of their promoter and enhancer regions.

The immediate-early (IE) gene of pseudorabies virus (PRV) has recently been sequenced for two virus strains. To investigate IE gene regulation and to examine the genome segment reported to encode latency-related transcripts in opposite polarity to the IE gene, sequence analysis has been extended by 5 kb from each end of the IE gene. The IE promoter (P1) was found to be more complex than previously recognized: it consisted of nine imperfect repeats, each containing five to six different consensus elements for transcription factor binding. A second promoter (P2) was discovered downstream of the IE gene. It contained numerous octamer consensus sequences (ATGCAAAT) and recognition sites for transcription factor Sp1; specific binding of nuclear proteins to four Sp1 sites was detected. An open reading frame (ORF3) bordering on P2 was identified, oriented antiparallel to the IE gene. Potential enhancer elements (E3 and E4) were isolated by the enhancer trap technique. Linked to P1 and a CAT indicator gene, E3 acted as an enhancer and E4 as a silencer. The PRV IE gene product repressed transcription from its own promoter and activated the SV40 early promoter. The transactivating virion protein Vmw65 of HSV1 had an opposite effect on these promoters.

Update and comparison of the immediate-early gene DNA sequences of two pseudorabies virus isolates.

Differences between the immediate-early gene DNA sequences of two pseudorabies virus isolates (Indiana-Funkhauser and Ka) were resolved and confirmed. The deduced amino acid sequences showed that regions 2 and 4 have fewer changes than the rest of the molecules. These two conserved regions may be functionally important.