Porcine deltacoronavirus accessory protein NS6 harnesses VPS35-mediated retrograde trafficking to facilitate efficient viral infection

ABSTRACT Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus with the potential to infect humans. Accessory protein NS6, encoded by PDCoV, is a key factor required for optimal viral replication. However, the precise mechanism(s) used by PDCoV NS6 to function remains largely unclear. The retromer is an evolutionarily highly conserved protein complex that plays an important role in normal cellular biological processes and viral replication. In this study, we identified VPS35, a subunit of the retromer complex, as a potential NS6-interacting protein via immunoprecipitation and mass spectrometry analysis in the context of PDCoV infection. Furthermore, we confirmed the interaction of VPS35 with NS6 by co-immunoprecipitation in both an overexpression system and PDCoV-infected cells. Knockdown of VPS35 by specific small interfering RNA inhibited wild-type PDCoV infection but did not suppress the infection of recombinant PDCoVs with an NS6 deletion, demonstrating that the VPS35-NS6 interaction is required for the proviral function of VPS35. We further demonstrated that the lysosomal pathway and Golgi trafficking are required for efficient PDCoV infection and that depletion of VPS35 prevented retrograde transport of PDCoV NS6 from endosomes to the Golgi, resulting in its lysosomal degradation and the inhibition of viral production. Taken together, our findings are the first to identify VPS35 as a critical host factor required for PDCoV to establish productive infection, revealing a novel evasion mechanism of PDCoV. IMPORTANCE Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has attracted considerable attention. However, whether retrograde transport is associated with PDCoV infection remains unclear. Our present study demonstrates that retromer protein VPS35 acts as a critical host factor that is required for PDCoV infection. Mechanically, VPS35 interacts with PDCoV NS6, mediating the retrograde transport of NS6 from endosomes to the Golgi and preventing it from lysosomal degradation. Recombinant PDCoVs with an NS6 deletion display resistance to VPS35 deficiency. Our work reveals a novel evasion mechanism of PDCoV that involves the manipulation of the retrograde transport pathway by VPS35, providing new insight into the mechanism of PDCoV infection. Retrograde transport has been reported to be closely associated with normal cellular biological processes and viral replication. As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has attracted considerable attention. However, whether retrograde transport is associated with PDCoV infection remains unclear. Our present study demonstrates that retromer protein VPS35 acts as a critical host factor that is required for PDCoV infection. Mechanically, VPS35 interacts with PDCoV NS6, mediating the retrograde transport of NS6 from endosomes to the Golgi and preventing it from lysosomal degradation. Recombinant PDCoVs with an NS6 deletion display resistance to VPS35 deficiency. Our work reveals a novel evasion mechanism of PDCoV that involves the manipulation of the retrograde transport pathway by VPS35, providing new insight into the mechanism of PDCoV infection.