Entry of sapelovirus into IPEC-J2 cells is dependent on caveolae-mediated endocytosis

Tingting Zhao,Xiuguo Hua,Xiaojuan Shen,Zhonghai Zhang,Xiangqian Yu,Li Cui

doi:10.1186/s12985-019-1144-6

Abstract

BackgroundPorcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, are a significant cause of enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. However, the life cycle of PSV on the molecular level is largely unknown.MethodsHere, we used chemical inhibitors, RNA interference, and overexpression of dominant negative (DN) mutant plasmids to verify the roles of distinct endocytic pathways involved in PSV entry into porcine small intestinal epithelial cell line (IPEC-J2).ResultsOur experiments indicated that PSV infection was inhibited when cells were pre-treated with NH4Cl or chloroquine. Inhibitors nystatin, methyl-β-cyclodextrin, dynasore and wortmannin dramatically reduced PSV entry efficiency, whereas the inhibitors chlorpromazine and EIPA had no effect. Furthermore, overexpression caveolin DN mutant and siRNA against caveolin also decreased virus titers and VP1 protein synthesis, whereas overexpression EPS15 DN mutant and siRNA against EPS15 did not reduce virus infection.ConclusionsOur findings suggest that PSV entry into IPEC-J2 cells depends on caveolae/lipid raft mediated-endocytosis, that is pH-dependent and requires dynamin and PI3K but is independent of clathrin and macropinocytosis.

Highlights

Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, are a significant cause of enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs
We addressed PSV entry into porcine small intestinal epithelial cell (IPEC-J2) by systematically perturbing the function of various cellular key factors involved in the known endocytic mechanisms using chemical inhibitors, Small interfering RNAs (siRNAs) silencing, and overexpression of dominant negative (DN) mutants of caveolin-1, Eps15 and dynamin-2
Our results suggested that PSV entry was caveolin, lipid raft, and dynamin-dependent and did not involve the clathrin and macropinocytosis pathway

Summary

Introduction

Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, are a significant cause of enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. Viruses generally enter cells via receptor-mediated endocytosis. The well-characterized clathrin-mediated endocytosis (CME) is the commonly used endocytic pathways for internalization of ligands, such as transferrin and epidermal growth factor [1], and many viruses, including type C foot-and-mouth disease virus [2] and echovirus 7 [3]. CME involves internalization of virus to be recruited to the plasma membrane through the formation of a clathrin coat [4]. Porcine sapelovirus (PSV) is a single-stranded, non-enveloped RNA virus, belonging to the genus of sapelovirus in the family Picornaviridae, and it is strongly associated with acute diarrhea, polioencephalomyelitis, pneumonia and reproductive disorders [13, 14]. Α2,3-linked sialic acid on GD1a as a PSV receptor in LLC-PK1 cells has been found [22], research on the mechanism underlying the pathogenesis, replication, and entry of PSV has not yet been well established

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