Ec CXCR4b influences RGNNV proliferation by interacting with the RGNNV capsid protein

Red-spotted grouper nervous necrosis virus (RGNNV) is an intensely virulent pathogen affecting cultured fish, capable of infecting various reared marine and freshwater species worldwide and causing severe financial losses in the aquaculture business. The capsid protein (CP) of RGNNV, as the viral surface’s sole structural protein, is essential for mediating RGNNV binding to susceptible host cells. However, the cellular receptors involved in RGNNV entry are still mostly unknown. In this study, immunoprecipitation-mass spectrometry (IP-MS) was employed to screen for candidate proteins that could interact with RGNNV-CP. The Epinephelus coioides CXC chemokine receptor 4b ( Ec CXCR4b) was found to be primarily localized on the cell membrane of grouper spleen (GS) cells and was identified as an interaction protein of RGNNV-CP by immunoprecipitation and immunofluorescence analysis. Furthermore, overexpression of Ec CXCR4b in host cells enhanced RGNNV attachment and internalization, whereas silencing Ec CXCR4b expression significantly inhibited replication of RGNNV and reduced viral entry. Additionally, treating cells with anti-CXCR4 antibodies to block cell surface Ec CXCR4b, or pretreating RGNNV virions with recombinant Ec CXCR4b protein effectively inhibited RGNNV binding to GS cells. In vivo, administering recombinant Ec CXCR4b protein significantly increased the survival rate of RGNNV-infected groupers. Moreover, exogenous overexpression of Ec CXCR4b facilitated RGNNV internalization in typically non-susceptible human lung adenocarcinoma cells (A549). Notably, we discovered that the clathrin endocytosis pathway was the mechanism by which Ec CXCR4b mediated RGNNV entrance into host cells. The results suggest that Ec CXCR4b is essential for RGNNV entry into host cells and could potentially serve as a therapeutic target for interventions against RGNNV infections. IMPORTANCE Red-spotted grouper nervous necrosis virus (RGNNV) is the most prevalent genotype in Betanodavirus and infects numerous fish species in warm water environments. In the viral infectious life cycle, virus-receptor recognition and interactions play a crucial role. Nevertheless, the host interaction proteins involved in RGNNV infection are still not completely understood. Therefore, further research is required to identify additional host proteins involved in RGNNV invasion to refine our understanding of its pathogenic mechanisms. In this study, we identified Epinephelus coioides CXC chemokine receptor 4b ( Ec CXCR4b) as RGNNV-CP interacting protein, and further analyses revealed that this interaction is of vital importance for RGNNV attachment and internalization. Additionally, Ec CXCR4b facilitates RGNNV entry into host cells via the clathrin-dependent endocytic mechanisms. These findings suggest that Ec CXCR4b serves as a critical host factor involved in RGNNV endocytosis, providing new insight into the intricate mechanisms underlying RGNNV entrance and useful prospective targets for antiviral medication development.