De novo design mini-binder proteins targeting the capsid-forming domain of type 2 PRRSV nucleocapsid protein to inhibit PRRSV replication in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses to swine industry but there is no effective antiviral drug. Nucleocapsid (N) protein is highly conserved in type 2 PRRSV and is considered as an important target for antiviral development. Mini-binders are novel protein drugs de novo designed for a specific protein target using a computational approach, which has great application prospects. Here, we employed a Rosetta-based approach to design mini-binders targeting the capsid-forming domain of N protein of type 2 PRRSV. The hydrophobic surface of the target protein was selected as the binding site and the promising binding modes were extensively explored. Three highly thermostable mini-binders with molecular weight of approximately 6 kDa were identified. All binders can penetrate the cell membrane and be distributed in the cytoplasm. Among them, binder 2 demonstrated robust anti-PRRSV efficacy in vitro, potently suppressing viral replication while exhibiting no interference with viral entry, internalization, or release. The binding between binder 2 and the capsid-forming domain of PRRSV N protein was confirmed by BLI assay and BiFC assay, indicating that the antiviral mechanism matched the expectation. Our study provides new insights into developing novel anti-PRRSV agents.