Nanobodies targeting SARS-CoV-2 papain-like protease exert dual antiviral and anti-inflammatory effects.

The emergence of SARS-CoV-2 variants and the rise of antiviral resistance necessitate the development of novel therapeutics targeting highly conserved viral proteins. The papain-like protease (PLpro) is a critical target due to its dual role in viral replication and immune evasion, particularly through the removal of ISG15 modifications from host proteins. However, nanobody-based strategies targeting PLpro for combined antiviral and anti-inflammatory purposes remain underdeveloped. This study reports the identification of two nanobodies, NbP1 and NbP2, that specifically disrupt the PLpro/ISG15 interaction interface. We characterized their binding specificity and affinity using yeast surface display and competitive fluorescence polarization assays. To ensure intracellular efficacy, the nanobodies were conjugated with cell-penetrating peptides (CPPs), resulting in significant inhibition of viral replication and the attenuation of inflammatory cytokine responses in both human colonic cells and a murine colitis model. Structural epitope mapping revealed that both nanobodies bind to key conserved residues within the PLpro/ISG15 and PLpro/ubiquitin interfaces. Our findings demonstrate that nanobodies targeting the PLpro/ISG15 interface can achieve synergistic antiviral and immunomodulatory effects, providing a proof-of-concept for a novel therapeutic approach to combat SARS-CoV-2 and potentially other emerging coronaviruses.