# Design and Characterization of an HRC-Derived Peptide Inhibitor of Canine Coronavirus Spike-Mediated Fusion

## TL;DR

This study designs a peptide from the HRC domain of canine coronavirus to block viral fusion and reduce infection, showing potential for therapeutic development.

## Contribution

The novel contribution is the design and characterization of an HRC-derived peptide inhibitor specific to canine coronavirus.

## Key findings

- The CCoV-HRC peptide showed no cytotoxicity at concentrations above 100 μM.
- The peptide effectively blocked CCoV-mediated membrane fusion and reduced viral infection in cell assays.
- In silico simulations supported the peptide's interaction with the S fusion core.

## Abstract

Canine coronavirus (CCoV), an alphacoronavirus belonging to the Coronaviridae family, is primarily associated with enteric infections in dogs. The ongoing evolution of coronaviruses through genetic recombination and mutation leads to the emergence of novel strains with increased pathogenicity, thereby raising the risk of cross-species transmission and spillover events. In this context, viral entry inhibitors represent a promising strategy, as they can serve as pivotal tools to prevent initial infection and subsequent viral replication. The S2 subunit of the spike (S) glycoprotein contains two heptad repeat regions (HRN and HRC), which play essential roles in the conformational changes required for viral fusion. In this study, we describe the design, synthesis, and functional evaluation of a peptide derived from the HRC domain of the CCoV S glycoprotein. First, we assessed the cytotoxicity of the CCoV-HRC peptide in two cell lines, HE293T and A72, and determined CC50 values > 100 μM. At non-toxic concentrations, the peptide effectively blocked membrane fusion mediated by the CCoV S glycoprotein and significantly reduced viral infection, as demonstrated both in cell–cell fusion assays and in live virus experiments. These findings were supported by in silico docking and molecular dynamics simulations, which provided structural insight into the interaction between CCoV-HRC and the S fusion core. Then, molecular analyses were conducted to evaluate the expression of the gene encoding the viral S protein, confirming the antiviral potential of CCoV-HRC peptide. Overall, these findings provide a solid foundation for the development of peptide-based therapeutics to treat or prevent CCoV infections.