Background: 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.

Results: 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.

Conclusion: 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.

Keywords: Autophagolysosomal degradation; BioPROTAC; Nanobody; Novel antiviral strategy; PRRSV.