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

Dóra Tombácz,Michael Snyder,Zsolt Boldogkői,Zsolt Balázs,Donald Sharon,Norbert Moldován,Attila Szűcs,Zsolt Csabai

doi:10.3389/fmicb.2017.01079

Dóra Tombácz, Michael Snyder + Show 6 more

Open Access

https://doi.org/10.3389/fmicb.2017.01079

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Abstract

In this study, we used the amplified isoform sequencing technique from Pacific Biosciences to characterize the poly(A)+ fraction of the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). Our analysis detected 34 formerly unidentified protein-coding genes, 10 non-coding RNAs, as well as 17 polycistronic and complex transcripts. This work also led us to identify many transcript isoforms, including 13 splice and 68 transcript end variants, as well as several transcript overlaps. Additionally, we determined previously unascertained transcriptional start and polyadenylation sites. We analyzed the transcriptional activity from the complementary DNA strand in five convergent HSV gene pairs with quantitative RT-PCR and detected antisense RNAs in each gene. This part of the study revealed an inverse correlation between the expressions of convergent partners. Our work adds new insights for understanding the complexity of the pervasive transcriptional overlaps by suggesting that there is a crosstalk between adjacent and distal genes through interaction between their transcription apparatuses. We also identified transcripts overlapping the HSV replication origins, which may indicate an interplay between the transcription and replication machineries. The relative abundance of HSV-1 transcripts has also been established by using a novel method based on the calculation of sequencing reads for the analysis.

Highlights

Herpes simplex virus type 1 (HSV-1) is a human pathogenic alphaherpesvirus from the Herpesviridae family
Many HSV-1 transcripts have been identified by Northern-blot analysis (Costa et al, 1984; Sedlackova et al, 2008), while the 5′ and 3′ termini of the transcripts have been determined by S1 nuclease mapping (McKnight, 1980; Rixon and Clements, 1982) or by primer extension (Perng et al, 2002; Naito et al, 2005) techniques
In many cases, the same transcription start sites (TSSs), transcription end sites (TESs), or splice junction were found in different transcripts detected within the same sequencing reaction, which further enhanced the number of independent Reads of Inserts (ROIs)

Summary

Introduction

Herpes simplex virus type 1 (HSV-1) is a human pathogenic alphaherpesvirus from the Herpesviridae family. Herpes is a lifelong infection, which often has mild or no symptoms. According to WHO’s first global estimates, worldwide more than 3.7 billion people under the age of fifty are infected with HSV-1 (Looker et al, 2015). The HSV-1 genome is composed of a unique long (UL) and a unique short (US) region, both being bracketed by inverted repeats (IRLs and IRSs, respectively). The HSV-1 DNA contains 89 protein-coding, 10 long non-coding (lnc)RNA genes (Rajcáni et al, 2004; McGeoch et al, 2006; Macdonald et al, 2012; Lim, 2013; Hu et al, 2016) and several micro RNAs (Du et al, 2015)

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