Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of the RhoA/ROCK/MLC Signaling Pathway

Tight junctions (TJs) are a major barrier and also an important portal of entry for different pathogens. Porcine sapovirus (PSaV) induces early disruption of the TJ integrity of polarized LLC-PK cells, allowing it to bind to the buried occludin coreceptors hidden beneath the TJs on the basolateral surface. However, the signaling pathways involved in the PSaV-induced TJ dissociation are not yet known. Here, we found that the RhoA/Rho-associated protein kinase (ROCK)/myosin light chain (MLC) signaling pathway was activated in polarized LLC-PK cells during the early infection of PSaV Cowden strain in the presence of bile acid. Specific inhibitors of RhoA, ROCK, and MLC restored PSaV-induced reduction of transepithelial resistance, increase of paracellular flux, intracellular translocation of occludin, and lateral membrane lipid diffusion. Moreover, each inhibitor significantly reduced PSaV replication, as evidenced by reductions in viral protein synthesis, genome copy number, and progeny viruses. The protein kinase C (PKC)/myosin light chain kinase (MLCK) and RhoA/ROCK/MLC phosphatase (MYPT) signaling pathways, which are known to dissociate TJs, were not activated during early PSaV infection. Among the aforementioned signaling pathways, the RhoA/ROCK/MLC signaling pathway was only activated by PSaV in the absence of bile acid, and specific inhibitors of this signaling pathway restored early TJ dissociation. Our findings demonstrate that PSaV binding to cell surface receptors activates the RhoA/ROCK/MLC signaling pathway, which in turn disrupts TJ integrity via contraction of the actomyosin ring. Our study contributes to understanding how PSaV enters the cells and will aid in developing efficient and affordable therapies against PSaV and other calicivirus infections. IMPORTANCE PSaV, one of the most important enteric pathogens, is known to disrupt TJ integrity to expose its buried coreceptor occludin in polarized LLC-PK cells. However, the cellular signaling pathways that facilitate TJ dissociation are not yet completely understood. Here, we demonstrate that early infection with PSaV in polarized LLC-PK cells in either the presence or absence of bile acids activates the RhoA/ROCK/MLC signaling pathway, whose inhibitors reverse the early PSaV infection-induced dissociation of TJs and reduce PSaV replication. However, early PSaV infection did not activate the PKC/MLCK and RhoA/ROCK/MYPT signaling pathways, which are also known to dissociate TJs. This study provides a better understanding of the mechanism involved in early PSaV infection-induced disruption of TJs, which is important for controlling or preventing PSaV and other calicivirus infections.