Biology subjects

Spatz, S. J.

Publications and source records attributed to Spatz, S. J..

4 recordsLinked to original sources

bioRxiv2026

Conserved herpesvirus protein kinase (CHPK)-mediated phosphorylation of viral proteins associated with nucleocytoplasmic trafficking during natural infection

The conserved herpesvirus protein kinase (CHPK) is encoded by all members of the Orthoherpesviridae and contributes to replication in cell culture but is not strictly required. Mareks disease virus (MDV) CHPK is dispensable for replication in cultured cells yet essential for horizontal transmission in chickens. To elucidate its role during natural infection, we performed RNA sequencing (RNA-seq) and mass spectrometry (MS)-based phosphoproteomics on spleen and feather follicle epithelial skin cells from chickens infected with wild-type or CHPK-null MDV. RNA-seq detected only a limited number of viral transcripts in the spleen--including latency-associated transcripts (LATs) and the major oncogene Meq--with minimal differences between wild-type and CHPK-null infections. In feather follicle epithelial skin cells, the full repertoire of viral genes was expressed, but only seven genes showed differential expression between wild-type and CHPK-null viruses. In striking contrast, MS-based phosphoproteomics identified many differentially phosphorylated proteins, including 21 viral proteins. These findings indicate that CHPKs critical functions in skin replication and subsequent horizontal transmission are primarily mediated through post-translational modifications (PTMs) rather than transcriptional regulation. Among the CHPK-targeted viral proteins were three MDV-unique proteins, eight conserved within the Alphaherpesvirinae, and ten conserved across the Orthoherpesviridae. In silico analysis revealed that many differentially phosphorylated serine and threonine residues lie near or within predicted nuclear localization signals (NLS) and nuclear export signals (NES). Functional validation confirmed that several of these motifs actively control nucleocytoplasmic shuttling of the respective viral proteins. Collectively, these data suggest that MDV CHPK orchestrates the subcellular localization of multiple viral proteins in epithelial skin cells via phosphorylation, thereby enabling efficient replication and horizontal transmission in the natural host. AUTHOR SUMMARYUnderstanding the mechanisms by which herpesviruses replicate and spread within their natural hosts and identifying the viral genes essential for these processes are fundamental to developing effective antiviral strategies. Mareks disease virus (MDV), a highly contagious alphaherpesvirus, remains a major economic threat to the global poultry industry while serving as a powerful natural animal model for studying herpesvirus pathogenesis and transmission in vivo. Using an established in vivo enrichment method for infected cells, we conducted a comprehensive analysis of viral gene expression, protein abundance, and post-translational modifications (PTMs) during natural infection. Remarkably, RNA sequencing revealed virtually no differences in viral transcription between wild-type and CHPK-null viruses in either spleen or feather follicle epithelial skin cells. In contrast, phosphoproteomics showed that CHPK extensively regulates the phosphorylation of multiple viral proteins specifically in skin epithelial cells. In silico and functional analyses further indicate that these CHPK-mediated phosphorylations occur near or within nuclear localization (NLS) and nuclear export (NES) signals, directly controlling the nucleocytoplasmic shuttling of key viral proteins. This work suggests CHPK as a master regulator of viral protein subcellular localization during replication in the natural host and highlights CHPK orthologs as promising broad-spectrum therapeutic targets against herpesviruses.

microbiology↗

bioRxiv2024

A functional interleukin-4 homolog is encoded in the genome of infectious laryngotracheitis virus: unveiling a novel virulence factor

Herpesviruses have evolved numerous immune evasion tactics, persisting within their hosts through self-perpetuating strategies. One such tactic involves acquiring functional copies of host genes encoding cytokines such as IL-6 (HHV-8), IL-10 (HHV-4, HHV-5), and IL-17 (SaHV-2). These viral mimics, or virokines, can bind to cellular receptors, modulating the natural cytokine signaling to manipulate the immune response in favor of the virus or stimulate target cell growth to enhance virus replication. In the course of full-length cDNA sequencing of infectious laryngotracheitis virus (ILTV) transcripts, a previously unknown highly-spliced gene was discovered in the viral genome predicted to encode a 147 amino acid protein with similarity to vertebrate interleukin-4. The three-intron gene structure was precisely conserved with chicken and other vertebrate IL-4 homologs, and the amino acid sequence displayed structural conservation with vertebrate homologs at the primary, secondary, and tertiary levels based on computational modeling. The viral IL-4 gene was subsequently identified in all sequenced ILTV genomes. The mature transcript was highly expressed both in vitro and in vivo, and protein expression in infected cells was confirmed using LC-MS/MS. Phylogenetic analyses, along with the conserved gene structure, suggested direct capture from a Galliformes host. Functionally, an LPS-stimulation assay showed that the expressed viral IL-4 homolog stimulated nitric oxide production in a macrophage cell line at comparable levels to recombinant chicken IL-4. A recombinant virus lacking vIL-4 exhibited slightly higher titers in cell culture compared to the parental strain. In vivo bird studies demonstrated reduced pathogenicity of the vIL-4 knockout compared to wildtype. These results represent the first report of a previously unknown virokine encoded in the ILTV genome expressing a functional IL-4 homolog and virulence factor. Author SummaryHerpesviruses are large DNA viruses, several of which express proteins that are homologous to host cytokines (termed virokines) and are thought to modulate the host immune response in favor of the virus. We report the identification of a novel virokine in the genome of infectious laryngotracheitis virus (ILTV) that is structurally and functionally similar to avian interleukin-4. The significance of this finding is threefold: O_LINovel mechanism: The identification of vIL-4 expands our understanding of the strategies employed by viruses to evade the host immune response, including through the acquisition, adaptation, and expression of cellular genes for their own advantage. C_LIO_LIImplications for disease pathogenesis: We demonstrate that vIL-4 plays a functional role in ILTV virulence. Understanding the mechanisms by which this happens could lead to the development of novel therapeutic strategies. C_LIO_LIEvolutionary insights: The presence of the highly spliced vIL-4 gene in the ILTV genome suggests direct genomic capture of a host gene, providing insights into the evolutionary history of ILTV and its interactions with avian theropods. C_LI Overall, this research represents a significant contribution to our understanding of viral immunology and has potential implications for the development of improved vaccines for ILTV infection.

microbiology↗

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↗