T-cell immunoglobulin and mucin domain 1 and transmembrane serine protease 2 synergistically enhance entry of a bat-derived merbecovirus.

Phosphatidylserine (PS) receptors facilitate entry of diverse enveloped viruses and have been implicated in enhancing coronavirus attachment, including for SARS-CoV-2, yet their precise contribution remains unclear. We examined this pathway using EjCoV-3, a recently identified bat merbecovirus. Exogenous PS, but not phosphatidylcholine, reduced viral infectivity in a concentration-dependent manner, suggesting competitive inhibition of receptor binding by virion-associated PS. T-cell immunoglobulin and mucin domain 1 (TIM-1) knockout (KO) in Vero/TMPRSS2 (transmembrane protease, serine 2) cells markedly reduced early viral infection. TIM-1 KO cells also showed markedly reduced virion attachment, indicating that TIM-1 acts as a PS-dependent attachment factor. Growth kinetics further revealed that TIM-1 contributes to efficient EjCoV-3 replication under conditions without exogenous protease and accelerates early viral growth in the presence of exogenous protease. A549 cells and A549/hACE2 cells are not susceptible to EjCoV-3 infection in the absence of exogenous proteases, and sustained viral replication was observed only when TIM-1 and TMPRSS2 were co-expressed, indicating that the two proteins synergistically promote infection. However, the expression of either factor alone was insufficient to sustain sustained viral replication. In contrast, in A549/EnACE2 cells, an EjCoV-3-susceptible cell line expressing a more compatible bat angiotensin-converting enzyme 2 (ACE2) orthologue, even in the absence of exogenous proteases, the expression of TMPRSS2 enhanced infection, whereas the expression of TIM-1 had no effect. These findings indicate that in environments where only low-affinity receptors are present, TIM-1 promotes EjCoV-3 infection primarily through PS-mediated attachment and functions in concert with TMPRSS2-dependent spike activation. This cooperative mechanism may reduce receptor barriers and enhance the potential for cross-species transmission of bat-derived coronaviruses, thereby contributing to their emergence as zoonotic pathogens.