Analysis of antiviral functions, differences and apoptotic effects of two novel MHC-Iα genotypes in orange-spotted grouper (Epinephelus coioides).

The Major Histocompatibility Complex (MHC) is a group of tightly linked, highly polymorphic genes that play a critical role in vertebrate immune responses. MHC is closely associated with various diseases, including autoimmune disorders, antigen invasion, and inflammation. Notably, MHC class I (MHC-I) molecules exhibit extensive polymorphism and have the ability to specifically bind to a diverse array of viral antigens, which is crucial for antigen presentation and antiviral immunity. However, studies on the differential functions of different MHC-I genotypes in response to fish viral diseases are still scarce. In our previous research, we cloned three novel MHC-Iα genotypes-EcMHC-IA∗01, EcMHC-IA∗02, and EcMHC-IA∗03 based on transcriptomic data from SGIV-infected grouper spleen cells. It has been established that the EcMHC-IA∗01 gene contributes to the resistance against SGIV infection. In this study, we investigated the differences in antiviral functions and apoptotic effects of EcMHC-IA∗02 and EcMHC-IA∗03. The full-length sequence homology of these two genotypes was 73.66 %, while the homology of the signal peptide was only 2.09 %. The polymorphism of domains may directly contribute to the functional differences among genotypes. Molecular characterization analysis revealed that EcMHC-IA∗02 and EcMHC-IA∗03 encoded 366-amino-acid and 357-amino-acid proteins, respectively, both containing typical domains of MHC class I molecules, including a signal peptide, MHC-I (α1, α2, α3) domains, and a transmembrane region. Based on their conserved structural features, domain organization, and overall sequence similarity to known classical MHC-I molecules in other species, these two genotypes are presumed to be classical MHC class I genes. Subcellular localization analysis showed that EcMHC-IA∗02 and EcMHC-IA∗03 were distributed throughout the cytoplasm. Functional studies demonstrated that overexpression of EcMHC-IA∗02 enhanced cytopathic effects (CPE), cell vacuolization, and the mRNA and protein expression of SGIV- and RGNNV-related genes, whereas EcMHC-IA∗03 overexpression produced the opposite effects, suppressing viral gene expression and cytopathology. Furthermore, siRNA-mediated knockdown of EcMHC-IA∗02 and EcMHC-IA∗03 reversed these phenotypes: silencing EcMHC-IA∗02 suppressed viral replication, while knockdown of EcMHC-IA∗03 enhanced it, further confirming their opposing roles in the regulation of SGIV and RGNNV replication. Meanwhile, qRT-PCR results showed that EcMHC-IA∗02 overexpression upregulated the transcription of pro-inflammatory cytokines (IL-6, IL-1β, and MDA5) but downregulated interferon signaling molecules (IRF-7, IFN-γ, and IFP-35) in vitro. In contrast, EcMHC-IA∗03 overexpression enhanced the transcription of both pro-inflammatory and interferon-related genes. Research on apoptosis found that EcMHC-IA∗02 and EcMHC-IA∗03 promoted apoptosis in FHM cells. Collectively, our findings highlight the distinct roles of EcMHC-IA∗02 and EcMHC-IA∗03 in grouper antiviral immunity. Studies have found that viruses can interfere with the signal peptide of MHC-Iα in mammals, reduce immune presentation, and achieve immune evasion. We speculate that in groupers, the virus utilizes the signal peptide domain of EcMHC-IA∗02 to achieve immune evasion and thus promote viral replication. However, differences in the domains do not prevent the two genotypes from exerting the same effect in promoting apoptosis. This work lays a foundation for further exploring genetic mechanisms underlying aquaculture species' antiviral defense.