Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus

Abstract

Pseudorabies virus (PRV) is an animal alphaherpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The ul54 gene of PRV and its homolog icp27 of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV ul54 function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the ul54 mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses.

Fig. 1

Deletion of the ul54 gene of PRV. Almost the entire ul54 gene was eliminated by a technique based on homologous recombination. A: this part of the figure shows the schematic representation of the inserted GFP expression cassette (illustrated at the top), as well as the knocked-out region of the PRV genome. B: Integrative Genomics Viewer (IGV) representation showing the presence of the mutation

Fig. 2

Growth curves of wt and mutant PRVs. This figure shows that abrogation of the ul54 function exerts a drastic effect on the viral growth. In the MOI experiment the mutant virus reached a lower concentration than the wt virus by 24 h p. i. The low MOI experiment revealed that the mutant virus exhibits a slower rate of growth and reaches a much lower titer by 24 hpi

Fig. 3

Cytopathic effect exerted by the wt and ul54-deleted viruses. High-magnification (200×) photomicrograph shows that – in contrast to the wt virus (A), which results in rounding of the infected cells – the shapes of the cells infected by the mutant virus exhibit normal phenotype (B) even after 24 hpi. Arrows show GFP expression from the cells infected by the ul54-KO virus

Fig. 4

The impact of the ul54 mutation on the expression of PRV genes. This plot shows the average R_r values of the three kinetic classes of PRV genes. The late genes are up-regulated at the early stage, while they are down-regulated at the late stage of infection in the mutant background. The early genes are down-regulated at 1h and 8h of infection in the ul54-KO virus. Black-filled circles with a straight line indicate the measured average R_r values of the E genes; white-filled triangles with a dashed line represent the E/L genes, while the values of L genes are labeled by black-filled squares with a dense dashed line

Fig. 5

The expression dynamics - based on the Rx values - of the four kinetic classes of PRV genes in the two examined genetic backgrounds. a. The expression of ie180 gene in the wt and mutant viruses. The major transactivator gene of PRV exhibits an inverse expression pattern in the two background. b. The average gene expression values of the E genes in the two viruses show inverted dynamics. c. The R_x values of the E/L genes indicate a complementary pattern of expression in the wt and mutant viruses. d. The gene expression dynamics of the L genes show the same expression pattern between the two viruses

Fig. 6

Comparisons of the expression patterns of transcription factor genes and their overlapping non-coding RNAs. A) Comparison of the expression kinetics of the ie180 and ep0 genes. The expression pattern of ie180 gene is similar, while the ep0 gene is complementary in the two genetic backgrounds. B) Comparison of the expression kinetics of ie180 and ep0 genes. The expression pattern of the two transcription factor genes is similar in the wt, while exhibits an inverse dynamics in the mutant virus. C) Comparison of the expression of ie180 and its antisense RNA genes. An inverse correlation can be observed between the transcription kinetics of IE180 and AST transcripts in the wt background, while no correlation exists between these transcripts in the mutant virus. D) Comparison of the expression of ep0 and its antisense RNA genes. An inverse correlation can be observed between the transcription kinetics of EP0 and LAT transcripts in the wt background, while these transcripts exhibit similar kinetics in the mutant virus. E) Comparison of the expression of LAT and AST transcripts. These antisense transcripts exhibit a similar expression pattern in the wt virus, while they express an inverse pattern in the mutant virus. F) The genomic location of ie180 and ep0 genes and their antisense RNAs

Fig. 6

Comparisons of the expression patterns of transcription factor genes and their overlapping non-coding RNAs. A) Comparison of the expression kinetics of the ie180 and ep0 genes. The expression pattern of ie180 gene is similar, while the ep0 gene is complementary in the two genetic backgrounds. B) Comparison of the expression kinetics of ie180 and ep0 genes. The expression pattern of the two transcription factor genes is similar in the wt, while exhibits an inverse dynamics in the mutant virus. C) Comparison of the expression of ie180 and its antisense RNA genes. An inverse correlation can be observed between the transcription kinetics of IE180 and AST transcripts in the wt background, while no correlation exists between these transcripts in the mutant virus. D) Comparison of the expression of ep0 and its antisense RNA genes. An inverse correlation can be observed between the transcription kinetics of EP0 and LAT transcripts in the wt background, while these transcripts exhibit similar kinetics in the mutant virus. E) Comparison of the expression of LAT and AST transcripts. These antisense transcripts exhibit a similar expression pattern in the wt virus, while they express an inverse pattern in the mutant virus. F) The genomic location of ie180 and ep0 genes and their antisense RNAs

Fig. 7

Replication of the PRV DNA in the wt and mutant viruses. The abrogation of ul54 gene leads to a significant reduction in DNA replication. These plots show the dynamics of DNA replication during the first 12 h pi (a) or between 1–24 h pi (b) in the mutant virus, as well as between 1–8h in the wt PRV

Fig. 8

Expression of CTO non-coding RNA in the wt and mutant viruses. CTO expression is significantly reduced (similar relative expression /R/ values in the first 2 h pi, a ~ two-fold difference at 4h pi, a ~ six-fold difference at 6h pi, while an almost eight-fold difference at 8h pi) in the mutant background, which may partly explain the shift in the initiation of DNA synthesis

Fig. 9

The dynamics of total gene expression from an individual PRV genome. This figure shows the relative copy numbers of the PRV genes in the two examined viruses, normalized to the DNA copy numbers. Similarly to DNA synthesis, the dynamics of global transcription are shifted by 2 hours