Full-Length Isoform Sequencing Reveals Novel Transcripts and Substantial Transcriptional Overlaps in a Herpesvirus

Abstract

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.

Long-Read Isoform Sequencing Reveals a Hidden Complexity of the Transcriptional Landscape of Herpes Simplex Virus Type 1

“…Additionally, as it has been detected in PRV (Tombácz et al, 2016), HSV transcripts also exhibit minor variations in both of their 5′ and 3′ termini.…”

“…Our earlier investigations revealed that several upstream genes of 3′-coterminal gene clusters of the PRV are also transcribed as monocistronic RNA molecules (Tombácz et al, 2016). However, in this study, with the exception of the transcripts expressed from the embedded genes, we only detected novel polycistronic molecules (Table 6) that include transcripts terminated upstream of the co-terminal PA sites.…”

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

“…We also characterised the novel transcripts reported in our recent publications1232. Supplementary Figure 1 shows the A6 values of these transcripts at different p.i.…”

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

“…PRV has a double-stranded linear DNA, composed of 143,423 bp with a mean GC content of 73.59% [16]. The PRV genome encodes 67 protein-coding and at least 20 non-coding RNA genes [17]. The transcriptome of the wild-type ( wt ) virus has been examined using various approaches, such as real-time RT-PCR [18] and microarray [19] analyses, as well as next generation [20] and third generation [17] sequencing platforms.…”

“…The transcriptome of the wild-type ( wt ) virus has been examined using various approaches, such as real-time RT-PCR [18] and microarray [19] analyses, as well as next generation [20] and third generation [17] sequencing platforms. It has recently been shown that almost the entire viral genome is transcriptionally active and many genes produce various transcript isoforms, including length and splice variants, as well as polycistronic transcription units with varying numbers of genes [17]. PRV genes, expressed in a cascade-like manner, are divided into three major temporal classes: immediate-early (IE), early (E) and late (L).…”