Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible coronavirus that has caused over 6 million fatalities. SARS-CoV-2 variants with spike mutations are frequently endowed with a strong capability to escape vaccine-elicited protection. Due to this characteristic, a broad-spectrum inhibitor against SARS-CoV-2 infection is urgently demanded. Ganoderma microsporum immunomodulatory protein (GMI) was previously reported to alleviate infection of SARS-CoV-2 through ACE2 downregulation whereas the impact of GMI on virus itself was less understood. Our study aims to determine the effects of GMI on SARS-CoV-2 pseudovirus and the more detailed mechanisms of GMI inhibition against SARS-CoV-2 pseudovirus infection.

ACE2-overexpressing HEK293T cells (HEK293T/ACE2) and SARS-CoV-2 pseudoviruses carrying spike variants were used to study the effects of GMI in vitro. Infectivity was evaluated by fluorescence microscopy and flow cytometry. Fusion rate mediated by SARS-CoV-2 spike protein was examined with split fluorescent protein /luciferase systems. The interactions of GMI with SARS-CoV-2 pseudovirus and ACE2 were investigated by immunoprecipitation and immunoblotting.

GMI broadly blocked SARS-CoV-2 infection in various cell lines. GMI effectively inhibited the infection of pseudotyped viruses carrying different emerged spike variants, including Delta and Omicron strains, on HEK293T/hACE2 cells. In cell-free virus infection, GMI dominantly impeded the binding of spike-bearing pseudotyped viruses to ACE2-expressing cells. In cell-to-cell fusion model, GMI could efficiently inhibit spike-mediated syncytium without the requirement of ACE2 downregulation.

GMI, an FDA-approved dietary ingredient, acts as a multifunctional broad-spectrum antiviral against SARS-CoV-2 and could become a promising candidate for preventing or treating SARS-CoV-2 associated diseases.