Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells

Abstract

The transcriptome of the Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) after primary latent infection of human blood (BEC), lymphatic (LEC) and immortalized (TIME) endothelial cells was analyzed using RNAseq, and compared to long-term latency in BCBL-1 lymphoma cells. Naturally expressed transcripts were obtained without artificial induction, and a comprehensive annotation of the KSHV genome was determined. A set of unique coding sequence (UCDS) features and a process to resolve overlapping transcripts were developed to accurately quantitate transcript levels from specific promoters. Similar patterns of KSHV expression were detected in BCBL-1 cells undergoing long-term latent infections and in primary latent infections of both BEC and LEC cultures. High expression levels of poly-adenylated nuclear (PAN) RNA and spliced and unspliced transcripts encoding the K12 Kaposin B/C complex and associated microRNA region were detected, with an elevated expression of a large set of lytic genes in all latently infected cultures. Quantitation of non-overlapping regions of transcripts across the complete KSHV genome enabled for the first time accurate evaluation of the KSHV transcriptome associated with viral latency in different cell types. Hierarchical clustering applied to a gene correlation matrix identified modules of co-regulated genes with similar correlation profiles, which corresponded with biological and functional similarities of the encoded gene products. Gene modules were differentially upregulated during latency in specific cell types indicating a role for cellular factors associated with differentiated and/or proliferative states of the host cell to influence viral gene expression.

Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication

“…Altering the growth conditions to allow cell migration and proliferation resulted in a substantial increase in the number of cells that were permissive for lytic infection. Similarly, within the background of many endothelial cells with long-term latent infections, spontaneous reactivation is observed in a small number of cells resulting in ORF59 expression (28,113) and virus replication (82). We have shown that this reactivation is characterized by coexpression of nuclear ORF59 and cytoplasmic LANA.…”

Native RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

“…Herpesviruses are adept viral pathogens that have co-evolved with their hosts over millions of years. Like all viruses their success is predicated on repurposing of the host transcriptional and translational machinery 1, 2 and through use of compact, gene-dense genomes with exceptional coding potential 3–7 . The 152 kb double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) includes roughly 80 genes, predominantly single exon open reading frames (ORFs), some transcribed as polycistronic mRNAs, along with a smaller number non-coding RNAs 8, 9 .…”

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

“…Just as NGS technologies are revolutionizing other fields, including molecular epidemiology, pathogen surveillance, and cancer biology, it is incumbent on the wider virology research community to embrace the most recent viral genome annotation/ reannotation projects (58)(59)(60)(61) and, most importantly, to incorporate the findings of new transcriptional profiling work into ongoing studies.…”