Effects of TRAF6 Knockout on Transcriptomic and Alternative Splicing Responses to VHSV Genotype II Infection

Viral hemorrhagic septicemia virus genotype II is generally considered less virulent than other genotypes. However, the molecular mechanisms underlying viral infection and its impact on host gene expression are poorly understood. The tumor necrosis factor receptor-associated factor 6 (TRAF6) is a pivotal molecule that is involved in different processes and plays an important role in the immune response during viral infection in fish. In this work, we investigated the effect of viral infection with VHSV genotype II on EPC and ΔTRAF6 cells at the gene expression level and on alternative splicing events (ASEs). Following VHSV genotype II infection, ΔTRAF6 cells exhibited more severe cytopathic effects accompanied by significantly higher viral copy numbers than EPC cells, indicating that TRAF6 plays a protective role in the antiviral response against VHSV genotype II. The transcriptomic analysis revealed that 1236 genes were differentially expressed in EPC cells, and 1372 in ΔTRAF6. Following viral infection, the immune-related pathways, including NOD-like receptor signaling, RIG-I-like receptor signaling, toll-like receptor signaling, cytokine-cytokine receptor signaling, C-type lectin receptor signaling pathway, and herpes simplex virus 1 infection, were activated in both cells, with lower gene ratios in ΔTRAF6 cells compared with EPC cells. Furthermore, TRAF6 influences alternative splicing in infected cells. Significant ASEs were observed in both cells, but were notably more frequent in ΔTRAF6 cells. Gene Ontology (GO) enrichment analysis and KEGG pathway enrichment analysis of differential alternative splicing genes showed enrichment in the mRNA surveillance and IgSF CAM signaling pathways. These results suggest that TRAF6 plays an important role in regulating the antiviral response at multiple levels, and that the absence of TRAF6 alters immune responses and signaling pathways, facilitating viral replication of VHSV genotype II and inducing cytopathic effects in cells. In addition, knocking out TRAF6 results in changes in alternative splicing correlating with weakened immune signaling and increased viral replication.