Luo X1, Guo L1, Zhang J1, Xu Y1, Gu W1, Feng L1, Wang Y2.

J Virol. 2017 Mar 8. pii: JVI.00202-17.

Abstract

Porcine epidemic diarrhea virus (PEDV), the causative agent of porcine epidemic diarrhea, has caused huge economic losses in pig-producing countries. Although it was long believed to replicate in the intestinal epithelium using aminopeptidase N as a receptor, the mechanisms of PEDV infection are not fully characterized. In this study, we found that PEDV infection of epithelial cells resulted in the disruption of the tight junctional distribution of occludin to the intracellular localization. Overexpression of occludin in target cells makes them more susceptible for PEDV infection, whereas ablation of occludin expression using siRNA in target cells significantly reduces their susceptibility to virus infection. However, the results observed with occludin siRNA indicate that occludin is not required for virus attachment. We conclude that occludin plays an essential role in PEDV infection at the post-binding stages. Furthermore, we observed that macropinocytosis inhibitors blocked occludin internalization and virus entry, indicating that virus entry and occludin internalization appear to be closely coupled. But the macropinocytosis inhibitors cannot impede virus replication once the virus has entered into host cells. These suggest that occludin internalization by macropinocytosis or a macropinocytosis-like process is involved in the virus entry events. Immunofluorescence confocal microscopy showed that PEDV was trapped at cellular junctional regions upon macropinocytosis inhibitor treatment, indicating that occludin may serve as a scaffold in the vicinity of virus entry. Collectively, these data show that occludin plays an essential role in PEDV infection at late entry events. Our observation may provide novel insights into PEDV infection and related pathogenesis.IMPORTANCE Tight junctions are highly specialized membrane domains whose main function is to attach adjacent cells together thereby forming intercellular seals. Here, we investigate for the first time the role of tight junction protein occludin in PEDV infection. We observed that PEDV infection induced the internalization of occludin. By using genetic modification methods, we demonstrate that occludin plays an essential role in PEDV infection. Moreover, PEDV entry and occludin internalization seem to be closely coupled. Our findings reveal a new mechanism of PEDV infection.