A direct RNA-seq-based EBV Latency Transcriptome Offers Insights into the Biogenesis of EBV Gene Products.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that establishes lifelong persistence in B cells. To reach this persistent state, EBV activates and drives proliferation of the infected B cell. In vitro, these EBV-infected cells can grow almost indefinitely as a lymphoblastoid cell line (LCL). EBV's transcription profile in LCLs (Latency III) produces six nuclear proteins (EBNAs), two latency membrane proteins (LMPs) and various non-coding RNAs, including microRNAs from the BHRF1 gene locus and the BamA-rightward transcripts (BARTs). The BART and EBNA transcription units are characterised by extensive alternative splicing. Here we have conducted a nanopore direct RNA-seq analysis of the LCL transcriptome of the widely used B95-8 BAC clone, including viruses with engineered variations (barcodes) in the first and last repeat of internal repeat 1 (IR1). Our pipeline ensures appropriate mapping of the W promoter (Wp) 5' exon, and corrects W1-W2 exon counts that misalign to IR1. Characterisation of EBNA reads revealed that transcripts using Wp more frequently encoded the EBNA-LP start codon than Cp transcripts; identified a novel splice donor in exon W2 and polyA site just before the EBNA2 start codon; provided insights into BHRF1 miRNA processing; and suggested co-ordination between polyA site usage and alternative splicing decisions, although improved read depth and integrity are required to confirm this. The BAC region disrupts the integrity of BART transcripts through premature polyadenylation and cryptic splice sites in the antisense of the hygromycin expression cassette. Finally, we identified a few extended reads that cross established gene boundaries, running from EBNA, to BART to LMP2 gene regions, including some novel exon usage between EBNA1 and the BART promoter that may have more relevance or abundance in other phases of the EBV lifecycle. We provide annotations based on these findings to enable better characterisation of the B95-8 BAC transcriptome.