Prediction of coronavirus 3C-like protease cleavage sites using machine-learning algorithms

The coronavirus 3C-like (3CL) protease, a cysteine protease, plays an important role in viral infection and immune escape. However, there is still a lack of effective tools for determining the cleavage sites of the 3CL protease. This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein, and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus, Betacoronavirus and Gammacoronavirus. Strong residue preferences were observed at the neighboring positions of the cleavage sites. A random forest (RF) model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes, and the model achieved an AUC of 0.96 in cross-validations. The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts. It achieved an AUC of 0.95 and predicted correctly 80% of the cleavage sites. Then, 1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model. These proteins were enriched in several GO terms related to the cytoskeleton, such as the microtubule, actin and tubulin. Finally, a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model. Overall, the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.