Epstein-Barr virus (EBV) and herpes simplex virus-1 (HSV-1) are members of the Herpesviridae family responsible for various human malignancies and acute infections. EBV is associated with a wide range of human cancers, including Burkitt's lymphoma, Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric cancer, while HSV-1 causes oral and genital lesions, encephalitis, and ocular herpes. Despite their clinical significance, effective antiviral treatments remain limited, and no approved vaccines are available. Here, we report the broad-spectrum antiviral activity of Ganoderma microsporum immunomodulatory protein (GMI), a safe dietary ingredient known for its immunomodulatory, anti-tumor, and antiviral properties.

A recombinant EBV strain (M81) carrying a GFP gene was used to assess the impact of GMI on EBV infection. EBV infectivity was evaluated using EBNA2 immunofluorescence staining for B cells, flow cytometry for GFP-positive HEK293 cells, and EBER in situ hybridization for AGS cells. Fusion mediated by EBV glycoproteins and the EphA2 receptor was detected using split GFP systems. The interaction between GMI and EBV proteins or receptors was analyzed through immunoprecipitation and immunoblotting. Similarly, HSV-1 strain 17 (HSV-Vp22-GFP) was used, and infectivity in HEK293T cells was assessed by flow cytometry for GFP-positive cells. Fusion via HSV-1 glycoproteins and the Nectin-1 receptor was detected with split fluorescence protein systems, and GMI interactions with HSV-1 proteins or receptors were analyzed by immunoprecipitation and immunoblotting. The AlphaFold 3 server was used to predict potential complex structures between GMI and EBV or HSV-1 gB.

GMI effectively inhibits EBV infection in epithelial cells in a dose-dependent manner by targeting both viral and host factors. Additionally, GMI exhibits antiviral activity against multiple EBV strains and significantly reduces EBV infection in primary B cells. Mechanistically, GMI interacts with EBV fusion glycoprotein gB and the host receptor EphA2, disrupting the viral fusion process necessary for infection. Given the structural conservation of gB among herpesviruses, we further tested GMI against HSV-1. Remarkably, GMI efficiently blocks HSV-1 infection by inhibiting both viral binding and fusion, with evidence of direct interaction with HSV-1 gB. In silico modeling suggests that GMI interacts with domain I of EBV and HSV-1 gB, potentially interfering with viral entry. The ability of GMI to inhibit both EBV and HSV-1 fusion highlights a possible shared antiviral mechanism that could extend to other herpesviruses.

Our findings provide the first evidence that GMI inhibits both EBV and HSV-1 infections by targeting the conserved gB-mediated fusion process. These results suggest that GMI could serve as a promising antiviral candidate against herpesviruses that rely on fusion-mediated entry.