Dóra Tombácz

Genetically timed, activity-sensor and rainbow transsynaptic viral tools

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

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

BackgroundPseudorabies 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.ResultsIn 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.ConclusionMost (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.

Full-length isoform sequencing reveals novel transcripts and substantial transcriptional overlaps in a herpesvirus

Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.

Characterization of pseudorabies virus transcriptome by Illumina sequencing

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.

Strain Kaplan of Pseudorabies Virus Genome Sequenced by PacBio Single-Molecule Real-Time Sequencing Technology

ABSTRACT Pseudorabies virus (PRV) is a neurotropic herpesvirus that causes Aujeszkys disease in pigs. PRV strains are widely used as transsynaptic tracers for mapping neural circuits. We present here the complete and fully annotated genome sequence of strain Kaplan of PRV, determined by Pacific Biosciences RSII long-read sequencing technology.

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.

Characterization of Novel Transcripts in Pseudorabies Virus

In this study we identified two 3′-coterminal RNA molecules in the pseudorabies virus. The highly abundant short transcript (CTO-S) proved to be encoded between the ul21 and ul22 genes in close vicinity of the replication origin (OriL) of the virus. The less abundant long RNA molecule (CTO-L) is a transcriptional readthrough product of the ul21 gene and overlaps OriL. These polyadenylated RNAs were characterized by ascertaining their nucleotide sequences with the Illumina HiScanSQ and Pacific Biosciences Real-Time (PacBio RSII) sequencing platforms and by analyzing their transcription kinetics through use of multi-time-point Real-Time RT-PCR and the PacBio RSII system. It emerged that transcription of the CTOs is fully dependent on the viral transactivator protein IE180 and CTO-S is not a microRNA precursor. We propose an interaction between the transcription and replication machineries at this genomic location, which might play an important role in the regulation of DNA synthesis.

Effects of deletion of the early protein 0 gene of pseudorabies virus on the overall viral gene expression.

Real-time RT-PCR analysis was applied to evaluate the impact of deletion of the early protein 0 (EP0) gene of pseudorabies virus (PRV) on the global expression of the viral transcripts during lytic infection in cultured porcine kidney cells. Our analysis showed that EP0 exerted an inhibitory effect on the transcription of the PRV genes in the early stage of infection, and alternating stimulatory and inhibitory effects on the viral gene expressions in the late stage of infection. The data also suggested that a general function of EP0 might be to reverse the kinetics of expression of early viral genes. We also observed that EP0 facilitated the development of correlations in the transcription kinetics between the immediate early 180 gene and the PRV transcripts, indicating that a major function of EP0 could be to modify the effects of the IE180 protein on the PRV transcriptome.

Herpesvirus-mediated delivery of a genetically encoded fluorescent ca 2+ sensor to canine cardiomyocytes

We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca2+ sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (Ito), kinetics of the intracellular Ca2+ transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the Ito current was significantly changed and no major shifts occurred in parameters of [Ca2+]i transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells.

Long-Read Sequencing of Human Cytomegalovirus Transcriptome Reveals RNA Isoforms Carrying Distinct Coding Potentials

The human cytomegalovirus (HCMV) is a ubiquitous, human pathogenic herpesvirus. The complete viral genome is transcriptionally active during infection; however, a large part of its transcriptome has yet to be annotated. In this work, we applied the amplified isoform sequencing technique from Pacific Biosciences to characterize the lytic transcriptome of HCMV strain Towne varS. We developed a pipeline for transcript annotation using long-read sequencing data. We identified 248 transcriptional start sites, 116 transcriptional termination sites and 80 splicing events. Using this information, we have annotated 291 previously undescribed or only partially annotated transcript isoforms, including eight novel antisense transcripts and their isoforms, as well as a novel transcript (RS2) in the short repeat region, partially antisense to RS1. Similarly to other organisms, we discovered a high transcriptional diversity in HCMV, with many transcripts only slightly differing from one another. Comparing our transcriptome profiling results to an earlier ribosome footprint analysis, we have concluded that the majority of the transcripts contain multiple translationally active ORFs, and also that most isoforms contain unique combinations of ORFs. Based on these results, we propose that one important function of this transcriptional diversity may be to provide a regulatory mechanism at the level of translation.