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

## TL;DR

The study shows how feline coronavirus uses intestinal enzymes to activate its spike protein and enter cells, highlighting a potential antiviral strategy.

## Contribution

The study identifies serine proteases as critical for feline coronavirus infection and reveals a compensatory cleavage mechanism at the S2′ site.

## Key findings

- Serine proteases like chymotrypsin, trypsin, and elastase enhance FCoV infection and syncytia formation.
- The S2′ cleavage site is crucial for spike activation and maintains infectivity even with mutations.
- Both free and membrane-bound serine proteases contribute to FCoV spike activation.

## Abstract

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.

Like many viruses, the common feline coronavirus (FCoV) relies on the host replication machinery to infect intestinal cells. Our study revealed how it works: FCoV exploits intestinal enzymes called serine proteases, which act like molecular scissors to cleave and activate the virus’s spike protein, the “key” the virus uses to unlock and enter cells. Without this activation step, infection cannot occur. The spike protein is cleaved by serine proteases primarily at two sites, S1/S2 and S2′, with S2′ playing a particularly critical role; even if mutations disable S2′, the virus cleverly uses backup sites nearby to remain infectious. Both free-floating serine proteases in the gut and membrane-bound ones on host cells can activate the spike, and blocking these enzymes significantly reduces viral infection. These findings reveal how FCoV takes advantage of its intestinal environment to ensure successful entry, and point to a promising antiviral strategy: targeting the host enzymes the virus depends on.