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.

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.

Comparison of immediate-early transcripts among bovine herpesvirus type 1 and type 5 strains differing in neurovirulent potential.

Northern (RNA) blot analysis was used to compare immediate-early (IE) transcripts among strains of bovine herpesvirus type 1 and type 5 differing in neurovirulent potential. Results obtained from the neurovirulent strain N569 were compared to those from two non-neurovirulent strains, Jura and K22. Strain N569 expressed three major IE transcripts with similar size and transcription pattern as previously reported for strain Jura and K22 (Wirth et al., 1989, 1991, 1992), except for some differences in the 5' terminal sequence common to the alternative spliced IE transcripts IER4.2 and IER2.9. More significant differences were discovered for some minor IE transcripts. Those transcribed over joined genome ends during replicative phase were found to be drastically overexpressed for strain N569 under IE condition. Among them two very long IE transcripts were detected clearly for strain N569, however hardly recognized for strain Jura and K22. These two transcripts were found to be encoded in addition to the joined terminal HindIII genome fragments by the second HindIII fragment J from the left genome end.

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.

Spatial and temporal distribution of bovine herpesvirus 1 transcripts.

Northern (RNA) blot analysis was used to determine the spatial and temporal distribution of bovine herpesvirus 1 (BHV-1) transcripts. Total RNA was isolated from Madin-Darby bovine kidney cells which had been infected with BHV-1.2b strain K22 or BHV-1.1 strain Jura in the presence or absence of metabolic inhibitors. Cloned restriction fragments representing the entire genome of strain K22 were labeled with 32P and hybridized to immobilized RNA. A total of 54 BHV-1 transcripts were found, ranging in size from 0.4 to larger than 8 kilobases (kb). The inverted repeat regions and an adjacent segment of the unique large part of the BHV-1 genome encoded three major immediate-early (IE) transcripts and one minor IE transcript enriched after cycloheximide treatment of infected cells. Late transcripts were identified by drastically reduced abundance after cytosine arabinoside (araC) treatment. Twelve late transcripts were encoded mainly by the unique long genome region, with a cluster of four transcripts located on HindIII fragment K (map units 0.677 to 0.733). The 21 transcripts unaffected by araC treatment were defined as early; they showed dispersed locations over the whole genome, with a cluster on the unique short sequence. The 17 remaining transcripts could not be classified unambiguously as early or late by these techniques. The IE transcript with a size of 4.2 kb exhibited homology with the single IE gene of pseudorabies virus, and the IE transcript with a size of 2.9 kb was encoded in part by the genome region known to be transcriptionally active during latency.