Deletion of the virion host shut-off gene of pseudorabies virus results in selective upregulation of the expression of early viral genes in the late stage of infection

AbstractA real-time RT-PCR technique was applied to evaluate the impact of deletion of the virion host shut-off (VHS) gene on the kinetics of pseudorabies virus gene expression. Selective suppression of early gene transcripts by the viral ribonuclease occurs after 4h of infection; while VHS protein appears to act non-selectively on the transcripts belonging in different kinetic classes in the first 2h of infection. VHS protein disrupts the close correlation between the transcription kinetics of the immediate-early 180 protein and the other pseudorabies virus transcripts. The typical pattern of early gene expression was found to be altered in the VHS gene-deleted virus in that the production rates of their transcripts did not decline from 4h post-infection. This observation led us to put forward the hypothesis that the VHS protein may play a pivotal role in the switch from the early to the late stage of infection

Dual Platform Long-Read RNA-Sequencing Dataset of the Human Cytomegalovirus Lytic Transcriptome

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Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay

<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo</it>. 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.</p> <p>Results</p> <p>In this study, we have developed and applied a quantitative reverse transcriptase-based real-time PCR technique in order to profile transcription from the whole genome of PRV after lytic infection in porcine kidney cells. We calculated the relative expression ratios in a novel way, which allowed us to compare different PRV genes with respect to their expression dynamics, and to divide the PRV genes into distinct kinetic classes. This is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qRT2-PCR. We additionally established the kinetic properties of uncharacterized PRV genes and revised or confirmed data on PRV genes earlier examined by traditional methods such as Northern blot analysis. Our investigations revealed that genes with the same expression properties form clusters on the PRV genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. Further, we detected inverse relationships as concerns the expressions of EP0 and IE180 mRNAs and their antisense partners.</p> <p>Conclusion</p> <p>Most (if not all) PRV genes begin to be expressed from the onset of viral expression. No sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral DNA synthesis. The expressions of the PRV genes were analyzed, categorized and compared by qRT2-PCR assay, with the average of the minimum cycle threshold used as a control for the calculation of a particular R value. In principle, this new calculation technique is applicable for the analysis of gene expression in all temporally changing genetic systems.</p

The effects of viral load on pseudorabies virus gene expression

<p>Abstract</p> <p>Background</p> <p>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 <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>, 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.</p> <p>Results</p> <p>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 (<it>ie180</it>) gene, was comparable to that of <it>ie180 </it>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.</p> <p>Conclusion</p> <p>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 <it>ie180 </it>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.</p

Characterization of the Dynamic Transcriptome of a Herpesvirus with Long-read Single Molecule Real-Time Sequencing

Herpesvirus gene expression is co-ordinately regulated and sequentially ordered during productive infection. The viral genes can be classified into three distinct kinetic groups: immediate-early, early, and late classes. In this study, a massively parallel sequencing technique that is based on PacBio Single Molecule Real-time sequencing platform, was used for quantifying the poly(A) fraction of the lytic transcriptome of pseudorabies virus (PRV) throughout a 12- hour interval of productive infection on PK-15 cells. Other approaches, including microarray, real-time RT-PCR and Illumina sequencing are capable of detecting only the aggregate transcriptional activity of particular genomic regions, but not individual herpesvirus transcripts. However, SMRT sequencing allows for a distinction between transcript isoforms, including length- and splice variants, as well as between overlapping polycistronic RNA molecules. The non-amplified Isoform Sequencing (Iso-Seq) method was used to analyse the kinetic properties of the lytic PRV transcripts and to then classify them accordingly. Additionally, the present study demonstrates the general utility of long-read sequencing for the time-course analysis of global gene expression in practically any organism