Nanobody-based bioPROTAC for viral protein degradation provides an antiviral strategy for porcine arterivirus

Proteolysis-targeting chimeras (PROTACs) are powerful tools for targeted protein degradation and are expected to contribute to a promising strategy for next-generation precision therapeutic antiviral drug development. Nanobody-based bioPROTACs can directly bind to protein and mediate target protein degradation, providing a potential antiviral strategy for RNA viruses featuring error-prone replication. Here, we aimed to establish a modular speckle-type POZ protein (SPOP)-derived bioPROTAC platform that enabled rapid antiviral drug construction through the substitution of a target protein-specific nanobody. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model pathogen, bioPROTACs molecules were successfully constructed by flexibly fusing nanobodies against PRRSV nonstructural protein 9 (Nsp9, viral RdRp) to the BTB domain of SPOP. BioPROTACs demonstrated specific degradation of target proteins in a dose-dependent manner, and a bivalent nanobody configuration enhanced the degradation efficiency to greater than 60%. BioPROTACs exhibited antiviral activity against multi-lineages of PRRSV and significantly potentiated the antiviral efficacy of non-neutralizing nanobodies in vitro. Furthermore, intravenous delivery of bioPROTAC-encoding constructs in mice achieved significant reduction of target protein levels within 24 h, demonstrating efficient in vivo degradation capability. Moreover, the combined administration of bioPROTACs via the mRNA-LNP system suppressed PRRSV proliferation and transmission in piglets, which was characterized by reduced viremia, alleviated lung damage, and a decrease in the piglet mortality rate to 25%. Importantly, we revealed that the subcellular localization of both the target protein and bioPROTACs determined the degradation pathway, confirming that cytoplasmic 9nb-SPOPΔNLS mediated Nsp9 degradation through the autophagy–lysosome pathway in the cytoplasm. This study expands the applicability of SPOP-derived bioPROTACs from nuclear proteins to cytoplasmic proteins, providing a novel strategy for developing antiviral therapies against highly variable viruses. The aim of the current study was to develop and validate modular bioPROTACs targeting essential viral proteins. We constructed the degraders by fusing target-specific nanobodies to the BTB domain of SPOP. More importantly, a combination of bioPROTACs targeting different stages of viral replication, delivered via mRNA-LNPs, suppressed viral replication in a pig model. These findings offer valuable insights into the target degradation mechanisms of SPOP-derived bioPROTACs and provide a foundation for the design of antivirals that have activity against multi-lineages of porcine arterivirus and overcome drug resistance.