BackgroundPseudorabies virus is a widely-studied model organism of the Herpesviridae family, with a compact genome arrangement of 72 known coding sequences. In order to obtain an up-to-date genetic map of the virus, a combination of RNA-sequencing approaches were applied, as recent advancements in high-throughput sequencing methods have provided a wealth of information on novel RNA species and transcript isoforms, revealing additional layers of transcriptome complexity in several viral species.ResultsThe total RNA content and polyadenylation landscape of pseudorabies virus were characterized for the first time at high coverage by Illumina high-throughput sequencing of cDNA samples collected during the lytic infectious cycle. As anticipated, nearly all of the viral genome was transcribed, with the exception of loci in the large internal and terminal repeats, and several small intergenic repetitive sequences. Our findings included a small novel polyadenylated non-coding RNA near an origin of replication, and the single-base resolution mapping of 3′ UTRs across the viral genome. Alternative polyadenylation sites were found in a number of genes and a novel alternative splice site was characterized in the ep0 gene, while previously known splicing events were confirmed, yielding no alternative splice isoforms. Additionally, we detected the active polyadenylation of transcripts earlier believed to be transcribed as part of polycistronic RNAs.ConclusionTo the best of our knowledge, the present work has furnished the highest-resolution transcriptome map of an alphaherpesvirus to date, and reveals further complexities of viral gene expression, with the identification of novel transcript boundaries, alternative splicing of the key transactivator EP0, and a highly abundant, novel non-coding RNA near the lytic replication origin. These advances provide a detailed genetic map of PRV for future research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0470-0) contains supplementary material, which is available to authorized users.

BackgroundPseudorabies virus (PRV), an alpha-herpesvirus of swine, is a widely used model organism in investigations of the molecular pathomechanisms of the herpesviruses. This work is the continuation of our earlier studies, in which we investigated the effect of the abrogation of gene function on the viral transcriptome by knocking out PRV genes playing roles in the coordination of global gene expression of the virus. In this study, we deleted the us1 gene encoding the ICP22, an important viral regulatory protein, and analyzed the changes in the expression of other PRV genes.ResultsA multi-timepoint real-time RT-PCR technique was applied to evaluate the impact of deletion of the PRV us1 gene on the overall transcription kinetics of viral genes. The mutation proved to exert a differential effect on the distinct kinetic classes of PRV genes at the various stages of lytic infection. In the us1 gene-deleted virus, all the kinetic classes of the genes were significantly down-regulated in the first hour of infection. After 2 to 6 h of infection, the late genes were severely suppressed, whereas the early genes were unaffected. In the late stage of infection, the early genes were selectively up-regulated. In the mutant virus, the transcription of the ie180 gene, the major coordinator of PRV gene expression, correlated closely with the transcription of other viral genes, a situation which was not found in the wild-type (wt) virus. A 4-h delay was observed in the commencement of DNA replication in the mutant virus as compared with the wt virus. The rate of transcription from a gene normalized to the relative copy number of the viral genome was observed to decline drastically following the initiation of DNA replication in both the wt and mutant backgrounds. Finally, the switch between the expressions of the early and late genes was demonstrated not to be controlled by DNA replication, as is widely believed, since the switch preceded the DNA replication.ConclusionsOur results show a strong dependence of PRV gene expression on the presence of functional us1 gene. ICP22 is shown to exert a differential effect on the distinct kinetic classes of PRV genes and to disrupt the close correlation between the transcription kinetics of ie180 and other PRV transcripts. Furthermore, DNA replication exerts a severe constraint on the viral transcription.

The pseudorabies virus (PRV; also known as Suid herpesvirus-1) is a neurotropic herpesvirus of swine. The us7 and us8 genes of this virus encode the glycoprotein I and E membrane proteins that form a heterodimer that is known to control cell-to-cell spread in tissue culture and in animals. In this study, we investigated the effect of the deletion of the PRV us7 and us8 genes on the genome-wide transcription and DNA replication using a multi-time-point quantitative reverse transcriptase-based real-time PCR technique. Abrogation of the us7/8 gene function was found to exert a drastic but differential effect on the expression of PRV genes during lytic infection. In the mutant virus, all kinetic classes of viral genes were significantly down-regulated at the first 6 h of infection, while having been upregulated later. The level of upregulation was the highest in the immediate-early (IE) and the early (E) genes; lower in the early-late (E/L) genes; and the lowest in the late (L) genes. The relative contribution of the L transcripts to the global transcriptome became lower, while the rest of the transcripts were expressed at a higher level in the mutant than in the wild-type virus.

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