TY - JOUR
T1 - Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host
AU - Volkening, Jeremy D.
AU - Spatz, Stephen J.
AU - Ponnuraj, Nagendraprabhu
AU - Akbar, Haji
AU - Arrington, Justine V.
AU - Vega-Rodriguez, Widaliz
AU - Jarosinski, Keith W.
N1 - Funding Information:
This report was supported by Agriculture and Food Research Initiative Competitive Grant nos. 2013-67015-26787, 2016-67015-26777, and 2020-67015-21399 from the USDA National Institute of Food and Agriculture to KWJ, and USDA-ARS NACA agreements nos. 58-6040-8-037 and 58-6040-0-015 to KWJ and SJS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank the Georgia Genomics and Bioinformatics Core, which provided the Illumina: Ribo-depleted RNA library preparation and NextSeq500 2×75bp sequencing service and the University of Illinois at Urbana-Champaign Proteomics Core for their guidance in LC-MS/MS services.
Publisher Copyright:
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
PY - 2023/6
Y1 - 2023/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85163920999&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85163920999&partnerID=8YFLogxK
U2 - 10.1371/journal.ppat.1011204
DO - 10.1371/journal.ppat.1011204
M3 - Article
C2 - 37289833
SN - 1553-7366
VL - 19
JO - PLoS pathogens
JF - PLoS pathogens
IS - 6
M1 - e1011204
ER -