Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host

Authors: VOLKENING, JEREMY - Base2bio; Spatz, Stephen; PONNURAJ, NAGENDRAPRABHU - University Of Illinois; AKBAR, HAJI - University Of Illinois; ARRINGTON, JUSTINE - University Of Illinois; VEGA-RODRIGUEZ, WIDALIZ - University Of Illinois; JARONSINSKI, KEITH - University Of Illinois

Submitted to: PLoS Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/29/2023
Publication Date: 6/8/2023
Citation: Volkening, J.D., Spatz, S.J., Ponnuraj, N., Akbar, H., Arrington, J.V., Vega-Rodriguez, W., Jaronsinski, K.W. 2023. Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host. PLoS Pathogens. 19(6):e1011204. https://doi.org/10.1371/journal.ppat.1011204.
DOI: https://doi.org/10.1371/journal.ppat.1011204

Interpretive Summary: Efficient transmission of viruses within a population is essential for dissemination. The virus-host relationship is relatively benign and has co-evolved over millions of years. The molecular mechanisms mediating transmission are complex. The role of the viral proteins involved in this process remain largely undefined. We have used Marek’s disease virus (MDV) tagged with a fluorescent marker as a model to investigate these proteins and their encoding RNA transcripts. The only site where cell-free MDV is produced in infected chickens is within feather follicle epithelial (FFE) skin cells. We used FFE cells enriched for actively replicating virus to measure in vivo both viral transcription and protein expression using combined RNA sequencing and liquid chromatography/mass spectrophotometry (generally referred to as LC/MS-MS bottom-up proteomics). We confirmed protein translation for 84 viral genes at high confidence and correlated relative protein abundance with RNA expression levels. Using this proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts in FFE and identified novel transcripts and peptides.

Technical Abstract: Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek’s disease is a devastating herpesviral disease of chickens caused by Marek’s disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5’ ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems.