Proteomic and Phosphoproteomic Analysis Reveals Differential Immune Response to Hirame Novirhabdovirus (HIRRV) Infection in the Flounder (Paralichthys olivaceus) under Different Temperature

SIMPLE SUMMARY: The outbreak of Hirame novirhabdovirus is significantly temperature dependent. The aim of this study was to identify differential responses of flounder to HIRRV infection at different temperatures by proteome and phosphoproteome. Post HIRRV infection under 10 °C and 20 °C, the enriched immune-related DEPs were both involved in RLR and NLR signaling pathways, apoptosis, phagosome and lysosome, and the DEPPs were also both enriched in spliceosome, mTOR signaling pathway and RNA transport. Compared with the group under 10 °C, the proteins and phosphoproteins involved in interferon production and signaling showed stronger response to infection under 20 °C. qRT-PCR assay showed that eight antiviral-related mRNA including IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44, MX1 and ISG15 displayed significantly stronger and quicker response at early infection under 20 °C. This study provided a comprehensive understanding of signaling alterations and differential antiviral responses of flounder to HIRRV infection under different temperatures. ABSTRACT: Hirame novirhabdovirus (HIRRV) is one of most serious viral pathogens causing significant economic losses to the flounder (Paralichthys olivaceus)-farming industry. Previous studies have shown that the outbreak of HIRRV is highly temperature-dependent, and revealed the viral replication was significantly affected by the antiviral response of flounders under different temperatures. In the present study, the proteome and phosphoproteome was used to analyze the different antiviral responses in the HIRRV-infected flounder under 10 °C and 20 °C. Post viral infection, 472 differentially expressed proteins (DEPs) were identified in the spleen of flounder under 10 °C, which related to NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, RNA transport and so on. Under 20 °C, 652 DEPs were identified and involved in focal adhesion, regulation of actin cytoskeleton, phagosome, NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway. Phosphoproteome analysis showed that 675 differentially expressed phosphoproteins (DEPPs) were identified in the viral infected spleen under 10 °C and significantly enriched in Spliceosome, signaling pathway, necroptosis and RNA transport. Under 20 °C, 1304 DEPPs were identified and significantly enriched to Proteasome, VEGF signaling pathway, apoptosis, Spliceosome, mTOR signaling pathway, mRNA surveillance pathway, and RNA transport. To be noted, the proteins and phosphoproteins involved in interferon production and signaling showed significant upregulations in the viral infected flounder under 20 °C compared with that under 10 °C. Furthermore, the temporal expression profiles of eight selected antiviral-related mRNA including IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44, MX1 and ISG15 were detected by qRT-PCR, which showed a significantly stronger response at early infection under 20 °C. These results provided fundamental resources for subsequent in-depth research on the HIRRV infection mechanism and the antiviral immunity of flounder, and also gives evidences for the high mortality of HIRRV-infected flounder under low temperature.