Serine protease-driven entry and S2 ′ cleavage flexibility of feline coronavirus during feline enterocyte infections

Coronaviruses not only hijack host cells to serve as viral factories but also exploit host proteolytic systems to activate their spike (S) protein, the key glycoprotein mediating receptor binding and membrane fusion. Feline coronavirus (FCoV), which initially replicates in the intestinal tract, has evolved to utilize local intestinal proteases for S protein activation. This activation occurs through proteolytic cleavage at specific regions on the S protein, known as cleavage sites (CSs). Two putative CSs have been proposed for FCoV: S1/S2 CS and S2' CS. Through a protease screen, we identified serine proteases as particularly critical for FCoV infection. Notably, three pancreatic serine proteases, chymotrypsin, trypsin, and elastase, enhanced FCoV infection and promoted syncytia formation despite their differing cleavage specificities, suggesting a flexible activation strategy. Furthermore, the membrane-bound serine proteases TMPRSS2 and TMPRSS11D also facilitated infection and syncytia formation in a strain-dependent manner. By analyzing the cleavage profiles of these serine proteases, we experimentally confirmed these two putative CSs on the FCoV S protein and identified additional CSs. Importantly, our analysis revealed a compensatory cleavage mechanism at the S2' CS that maintains spike activation even when mutations disrupt the canonical cleavage motif, underscoring the central role of S2' CS in viral infection. Additionally, an acidic microenvironment is required for efficient infection. Together, these findings illustrate how FCoV adapts to locally available serine proteases to optimize S protein priming and intestinal cell entry.