Biology subjects

Jarosinski, K. W.

Publications and source records attributed to Jarosinski, K. W..

2 recordsLinked to original sources

bioRxiv2023

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

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 their close relationship to their natural host. Mareks disease is a devastating herpesviral disease of chickens caused by Mareks disease virus (MDV) and an excellent natural model to study skin- tropic herpesviruses and transmission. Similar to varicella zoster virus that causes chicken pox in humans, the only site where fully productive replication occurs is in epithelial skin cells and this is required for host to host transmission. Here, we enriched for actively replicating virus in feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined 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 both intron-spanning sequencing reads as well as 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 evidence of 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. Author SummaryIn the natural host, the transcriptome and proteome of many herpesviruses are poorly defined. Here, we evaluated the viral transcriptome and proteome in feather follicle epithelial skin cells of chickens infected with Mareks disease virus (MDV), an important poultry pathogen as well as an excellent model for skin-tropic human alphaherpesvirus replication in skin cells. Using fluorescently tagged virus, we significantly enriched the number of infected cells sampled from live chickens, greatly enhancing the detection of viral transcripts and proteins within a host background. Based on this, we could confirm the translation of most transcripts using deep MS/MS-based proteomics and identify novel expressed peptides supportive of an increasingly complex translational and regulatory viral landscape. The demonstrated deep peptide sequencing capability can serve as a template for future work in herpesviral proteomics.

microbiology↗

bioRxiv2022

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK)

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Mareks disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK--particularly its kinase activity--is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection. Author SummaryMareks disease herpesvirus (MDV) is an important pathogen in the poultry industry. Current vaccines reduce disease but do not protect chickens from infection. Understanding natural infection provides vital information for developing potential therapies to protect against Mareks disease, while also providing a robust natural virus-host model to study herpesvirus pathogenesis. The conserved Herpesviridae protein kinase (CHPK) of MDV is essential for the initiation of natural infection, and we identified the virion protein US10 as a potential target for CHPK during natural infection. Our results showed that MDV CHPK was required for expression of pUS10 in both cell culture and in chickens. Although MDV lacking pUS10 replicated and spread in experimentally infected chickens, it was less virulent implicating an important role of pUS10 in natural infection of the host. These results emphasize the importance of studying specific viral proteins and their functions during infection of the natural host.

microbiology↗