Calpain-1: a Novel Antiviral Host Factor Identified in Porcine Small Intestinal Mucus

The thick mucus layer covering of the intestinal epithelium has received increasing attention, owing to its protective role in intestinal infection. However, the exact mechanisms by which the mucus increases intestinal resistance against viral infection remain largely unclear. Here, we identify prominent antiviral activity of the small intestinal mucus and extracted total mucus proteins, as evidenced by their inhibitory effects against porcine epidemic diarrhea virus (PEDV) infection. Of all the extracted mucus proteins, mucin 2 and fraction III (~70 kDa) exhibited potent antiviral activity. We further evaluated the antiviral effects of three candidate factors in fraction III and found that calpain-1 contributed substantially to its antiviral activity. In vivo studies demonstrated that oral administration of calpain-1 provided effective protection against intestinal PEDV infection. As a calcium-activated cysteine protease, calpain-1 inhibited viral invasion by binding to and hydrolyzing the S1 domain of the viral spike protein. The region between amino acids 297 and 337 in the b domain of PEDV S1 protein was critical for calpain-1-mediated hydrolysis. Further investigation indicated that calpain-1 could be produced by goblet cells between intestinal epithelia. Taken together, the results of our study revealed calpain-1 to be a novel antiviral protein in porcine small intestinal mucus, suggesting that calpain-1 has potential for defending against intestinal infections. IMPORTANCE Although the antiviral activity of the intestinal mucus was recognized 20 years ago, the antiviral active ingredients in the mucus are poorly understood. Currently, most research on antiviral molecules in the intestinal mucus remains limited to members of the mucin family. This study identified the cysteine protease calpain-1 as a novel antiviral protein in porcine small intestinal mucus and revealed its underlying protective mechanism for the first time. This mechanism involves inhibiting porcine epidemic diarrhea virus (PEDV) invasion by binding and hydrolyzing the S1 domain of the viral spike protein. Furthermore, the results of our PEDV-challenge experiment in piglets indicated that calpain-1 provides effective protection against intestinal PEDV infection. Our findings provide new insights into the protective function of the small intestinal mucus. In addition to potential therapeutic implications for the swine industry, our analysis of antiviral proteins in the small intestinal mucus may have implications for the prevention and control of coronavirus infection in humans.