Effects of deletion of the early protein 0 gene of pseudorabies virus on the overall viral gene expression (original) (raw)
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Veterinary Microbiology, 1998
Pseudorabies virus (PRV) early protein 0 (EP0) functions as a transactivator of the viral gene promoters. In transient expression assays employing chloramphenicol acetyl transferase (CAT) reporter constructs, EP0 and the immediate-early protein IE180 act in an additive manner to activate transcription from the thymidine kinase (TK) and glycoprotein G (gG) gene promoters. EP0 enhanced the synthesis of infectious virus in cotransfection experiments with the EP0-expression plasmid and PRV genomic DNA. EP0 was detected by Western blot analysis in the purified virions. These results may indicate that EP0 in the virions acts as an important transactivator to express the immediate-early gene efficiently in the first stage of infection, and IE180 and EP0 expressed after the infection cooperatively activate the early and late gene expression in the later stage of infection.
Virus genes, 2017
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. T...
The effects of viral load on pseudorabies virus gene expression
BMC Microbiology, 2010
Background: Herpesvirus genes are classified into distinct kinetic groups on the basis of their expression dynamics during lytic growth of the virus in cultured cells at a high, typically 10 plaque-forming units/cell multiplicity of infection (MOI). It has been shown that both the host response and the success of a pathogen are dependent on the quantity of particles infecting an organism. This work is a continuation of an earlier study , in which we characterized the overall expression of PRV genes following low-MOI infection. In the present study, we have addressed the question of whether viral gene expressions are dependent on the multiplicity of infection by comparing gene expressions under low and high-MOI conditions. Results: In the present study, using a real-time RT-PCR assay, we address the question of whether the expression properties of the pseudorabies virus (PRV) genes are dependent on the number of virion particles infecting a single cell in a culture. Our analysis revealed a significant dependence of the gene expression on the MOI in most of these genes. Specifically, we found that most of the examined viral genes were expressed at a lower level at a low MOI (0.1) than at a high MOI (10) experiment in the early stage of infection; however, this trend reversed by six hour post-infection in more than half of the genes. Furthermore, in the high-MOI infection, several PRV genes substantially declined within the 4 to 6-h infection period, which was not the case in the low-MOI infection. In the low-MOI infection, the level of antisense transcript (AST), transcribed from the antiparallel DNA strand of the immediate-early 180 (ie180) gene, was comparable to that of ie180 mRNA, while in the high-MOI experiment (despite the 10 times higher copy number of the viral genome in the infected cells) the amount of AST dropped by more than two log values at the early phase of infection. Furthermore, our analysis suggests that adjacent PRV genes are under a common regulation. This is the first report on the effect of the multiplicity of infection on genome-wide gene expression of large DNA viruses, including herpesviruses. Conclusion: Our results show a strong dependence of the global expression of PRV genes on the MOI. Furthermore, our data indicate a strong interrelation between the expressions of ie180 mRNA and AST, which determines the expression properties of the herpesvirus genome and possibly the replication strategy (lytic or latent infection) of the virus in certain cell types.
BMC Genomics, 2008
Background: Transcriptomic approaches are relevant for studying virus-host cell dialogues to better understand the physiopathology of infection and the immune response at the cellular level. Pseudorabies virus (PrV), a porcine Alphaherpesvirus, is a good model for such studies in pig. Since PrV displays a strong tropism for mucous epithelial cells, we developed a kinetics study of PrV infection in the porcine PK15 epithelial cell line. To identify as completely as possible, viral and cellular genes regulated during infection, we simultaneously analyzed PrV and cellular transcriptome modifications using two microarrays i.e. a laboratory-made combined SLA/PrV microarray, consisting of probes for all PrV genes and for porcine genes contained in the Swine Leukocyte Antigen (SLA) complex, and the porcine generic Qiagen-NRSP8 oligonucleotide microarray. We confirmed the differential expression of a selected set of genes by qRT-PCR and flow cytometry.
Veterinary Research, 2006
Pseudorabies virus (PRV) is an alpha herpesvirus that causes Aujezsky disease in the pig. To characterize the impact of PRV infection on cellular expression, we used microarrays consisting of 9850 oligonucleotides corresponding to human genes and examined the expression levels of mRNA isolated 0.5, 3, 6, and 9 h post infection (hpi) from cultures of infected HEK-293 cells. Very few changes were observed during the first 3 h of infection but significant modifications in the cell expression of more than 1000 genes were clearly apparent by 6 hpi. More than 2400 genes were either up-or down-regulated during the 9 h experiment. These results were then analyzed using gene ontology and the MAPP and MAPPFinder software. This comprehensive analysis clearly shows that the down-regulated genes were mainly involved in macromolecular synthesis (DNA, RNA and proteins) and the cell cycle. The up-regulated genes primarily concerned the regulation of DNA transcription, developmental processes (central nervous system development, neurogenesis, angiogenesis), cell adhesion and potassium transport. This study is the first qualitative analysis of a gene expression survey in a human cell line following PRV infection. It demonstrates global changes in the cell expression profile, and identifies the main biological processes that are altered during virus replication.
Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay
BMC Genomics, 2009
Background: Pseudorabies virus (PRV), a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection.
Archives of virology, 2010
The pseudorabies virus (PRV) glycoprotein known as gG is generally regarded as an early protein, and the immediate early IE180 protein regulates its expression during infection. This study, however, provides evidence that although induction by IE180 is observed, the expression of a marker protein (EGFP), or gG itself, under the control of the gG promoter, can also occur independently of the expression of IE180. This result was demonstrated both with transient transfection assays using plasmids and with viral infections. In transient transfections, the expression under control of the gG promoter depends on the cell type and surprisingly, can be 1.3-fold higher than the expression under the control of the IE180 promoter in Hela Tet-Off cells. Recombinant PRV S3 was constructed by replacing gE in the PRV genome with a chimeric transgene, expressing EGFP under the control of the gG promoter. In PK15 cells infected with NIA-3 wild-type virus or with S3 recombinant virus, expression of gG PRV mRNA (or EGFP mRNA) under the control of the gG promoter in the presence of cycloheximide was detected by RT-PCR. This again indicates that some basal expression was produced in infected cells independently of IE180. This expression was augmented by IE180 protein in both plasmid transfections and viral infections.
Virus Genes, 2004
The virion host shutoff (vhs) protein is a virion component of Alphaherpesviruses, including pseudorabies virus. In this work, the upstream sequences of vhs gene of pseudorabies virus (TNL strain) was cloned and sequenced. We linked the upstream sequences of vhs gene to the CAT reporter gene and examined the promoter function of this region. The immediate-early protein IE180 of Pseudorabies Virus (PRV) is expressed immediately after infection and plays a vital role in the regulation of other viral genes. Our results demonstrated that the vhs promoter was regulated by the IE180 in a dosagedependent manner; the vhs promoter was stimulated by low concentration of IE180 but suppressed by high concentration of IE180. Mutational analysis indicated that the only IE180 binding site at the vhs promoter was not essential for its function; however, a Sp1 binding site (15 bp downstream to TATA box) was critical to its function. In addition, the result of cotransfection demonstrated that early protein 0 (EP0) of PRV, another protein with transcriptional function, inhibited the activity of the vhs promoter.