Introduction
Atlantic salmon (Salmo salar) is one of the most globally economically important marine aquaculture species. Improving our understanding of the teleost immune response to pathogens could help combat the high disease burden in the aquaculture sector. Ubiquitination is an essential post-translational modification , known to play critical roles in the initiation, regulation, and termination of the innate immune system in mammalian species. Ubiquitin is present in all eukaryotic cells and is highly evolutionary conserved suggesting that its role in immunity may be conserved in other vertebrates. Despite evidence of many virus-inducible ubiquitination-related genes in fish4 and the observed up-regulation of the ubiquitination pathway in infected fish5, the role of ubiquitination in response to infection is still poorly understood. To identify the network of ubiquitin-related responses to viral infection this study combine s analysis of the ubiquitinated proteome and gene expression of an Atlantic salmon cell line post-viral infection , using mass spectroscopy and RNA sequencing .
Materials and Methods
Salmon Head Kidney cells (SHK-1) were inoculated with Infectious salmon anaemia virus (ISAV) or Infectious pancreatic necrosis virus (IPNV). Samples were collected at 24 and 48 hours post-infection (hpi), along with time-matched controls (4 biological replicates per condition ). For proteomics, c ells were ly sed by freeze-thaw and ubiquitinated proteins were enriched using HaloTagged Ubiquilin. Ubiquitinated protein content was measured via western blot, then submitted for Mass spectroscopy analysis. Mass spectroscopy was performed with data-dependent scanning , selection based on the host and viral proteome. RNA was extracted using TRIzol, and polyA RNA-seq libraries prepared using standard Illumina protocols and sequenced on a Novaseq 6000 as 150PE reads. Low-quality reads were removed, and gene expression was estimated using Kallisto6 and the Atlantic salmon reference transcriptome ( Ssal_v3.1, GCA_905237065.2)7.
Results
Infection of SHK-1 cells with ISAV induced a global up- regulation of ubiquitin, whilst IPNV infection induced an overall decrease in cellular ubiquitin (Figure 1A) . This is consistent with the mass spectroscopy data, where the total ubiquitination profile of ISAV samples incurred a net positive fold change , whilst IPNV induced an overall negative fold change in ubiquitinated proteins , especially at 24 hpi (Figure 1B). A small number of proteins (4 -50 depending on the condition) showed significant differences in ubiquitination in response to infection, including several relevant immune genes. For example , a TRIM ubiquitin ligase, TRIM25-like protein, showed significantly increased ubiquitination in response to ISAV at 24 hpi . Interestingly, all viral proteins were identified in the mass spectrometry data, and both the viral polymerase basic protein 2 and Matrix Protein 1 of ISAV contained di-glycine remnants consistent with ubiquitination.
RNA sequencing of the same samples revealed a massive up-regulation of gene expression in response to IPNV, both at 24 hpi and 48 hpi, while the response to ISAV was considerably more restricted (1033 vs 157 differentially expressed genes at 24 hpi).
Conclusion
Our results demonstrate a differential regulation of ubiquitination upon viral infection of different viruses in Atlantic salmon cells. We have found a TRIM25-like protein amongst the proteins with increased ubiquitination in response to ISAV. However, IPNV infection mainly led to decreased levels of ubiquitination in a small number of proteins. This lack of ubiquitination response contrasts with the large dysregulation of gene expression, and may suggest IPNV has the ability to regulate ubiquitination to enable infection. Additionally, multiple viral proteins contained ubiquitination sites upon digestion, namely ISAV viral polymerase basic protein 2 and matrix protein 1.
References
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