Hybrid sequencing discloses unique aspects of the transcriptomic architecture in equid alphaherpesvirus 1

Abstract

This study employed both short-read sequencing (SRS, Illumina) and long-read sequencing (LRS Oxford Nanopore Technologies) platforms to conduct a comprehensive analysis of the equid alphaherpesvirus 1 (EHV-1) transcriptome. The study involved the annotation of canonical mRNAs and their transcript variants, encompassing transcription start site (TSS) and transcription end site (TES) isoforms, in addition to alternative splicing forms. Furthermore, the study revealed the presence of numerous non-coding RNA (ncRNA) molecules, including intergenic and antisense transcripts, produced by EHV-1. An intriguing finding was the abundant production of chimeric transcripts, some of which potentially encode fusion polypeptides. Moreover, EHV-1 exhibited a greater incidence of transcriptional overlaps and splicing compared to related viruses. It is noteworthy that many genes have their unique TESs along with the co-terminal transcription ends, a characteristic scarcely seen in other alphaherpesviruses. The study also identified transcripts that overlap the replication origins of the virus. Moreover, a novel ncRNA, referred to as NOIR, was found to intersect with the 5'-ends of longer transcript isoform specified by the major transactivator genes ORF64 and ORF65, surrounding the OriL. These findings together imply the existence of a key regulatory mechanism that governs both transcription and replication through, among others, a process that involves interference between the DNA and RNA synthesis machineries.

Keywords: Direct RNA sequencing; EHV-1; Equid alphaherpesvirus 1; Illumina sequencing; Long-read sequencing; Nanopore sequencing; Replication origin; Transcriptome.

Fig. 1

Workflow. This figure shows the steps of our analysis starting from the infection of RK-13 cells with a field isolate of EHV-1 and ends with the annotation of transcripts.

Fig. 2

Canonical EHV-1 transcripts. This figure presents the canonical transcripts of EHV-1 identified using the LoRTIA program suit. Canonical mRNAs are defined as the most abundant transcript containing the same ORFs and the same exon-intron structure if spliced. All of the annotated non-coding RNAs and fusion transcripts are also depicted. Color code: black: mRNAs, red: ncRNAs, blue: fusion transcripts, yellow: mRNAs undetected by dRNA-Seq and fusion transcripts of which only the introns and TESs were detected by dRNA-Seq but not the TSS of the transcript. The shade of the colors corresponds to the abundance: 1: 1–9 reads, 2: 10–49 reads, 3: 50–199 reads, 4: 200–999 reads, 5: >1000 reads.

Fig. 3

TATA boxes and poly(A) signals (A) Genomic surrounding of TSSs with TATA box within a ±5 bp interval. The first letter of TSSs (position 0) is enriched with G/A bases, while the − 1 position contains mainly C/T bases. (B) A) Genomic surrounding of TSSs without TATA box within a ±5 bp interval. The 0 and + 1 TSS positions are enriched with G letters (C) Sequence motifs of transcripts containing polyadenylation signals. (D) Sequence motifs of transcripts without polyadenylation signals.

Fig. 4

Transcription start and end sites. A. Genome-wide localization of TSSs. The relative amount of the TSSs is calculated from a mixed time-point sample. B. Genome-wide localization of TESs. The relative amount of the TSSs is calculated from a mixed time-point sample. C. TSS clusters illustrated by 5 examples D. TES clusters illustrated by 5 examples.

Fig. 5

Transcription near the replication origins. In this figure, the presence of a transcript in the dRNA-Seq data was a prerequisite. A. OriS: ORF64-65 region. B. OriL: ORF35-41 region. The color code is defined in the figure. The shade of the colors corresponds to the abundance: 1: 1–9 reads, 2: 10–49 reads, 3: 50–199 reads, 4: 200–999 reads, 5: >1000 reads.

Fig. 6

Splicing and fusion transcripts. In this figure, the presence of a transcript in the dRNA-Seq data was a prerequisite. A. ORF6-12. B. ORF35-38. C. ORF44-50. D. ORF53-58. The color code is defined in the figure. The shade of the colors corresponds to the abundance: 1: 1–9 reads, 2: 10–49 reads, 3: 50–199 reads, 4: 200–999 reads, 5: >1000 reads.

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