title: Cell entry of avian reovirus follows a caveolin-1-mediated and dynamin-2-dependent endocytic pathway that requires activation of p38 mitogen-activated protein kinase (MAPK) and Src signaling pathways as well as microtubules and small GTPase Rab5 protein.
abstract: Very little is known about the mechanism of cell entry of avian reovirus (ARV). The aim of this study was to explore the mechanism of ARV entry and subsequent infection. Cholesterol mainly affected the early steps of the ARV life cycle, because the presence of cholesterol before and during viral adsorption greatly blocked ARV infectivity. Although we have demonstrated that ARV facilitating p38 MAPK is beneficial for virus replication, its mechanism remains unknown. Here, we show that ARV-induced phosphorylation of caveolin-1 (Tyr(14)), dynamin-2 expression, and Rac1 activation through activation of p38 MAPK and Src in the early stage of the virus life cycle is beneficial for virus entry and productive infection. The strong inhibition by dynasore, a specific inhibitor of dynamin-2, and depletion of endogenous caveolin-1 or dynamin-2 by siRNAs as well as the caveolin-1 colocalization study implicate caveolin-1-mediated and dynamin-2-dependent endocytosis as a significant avenue of ARV entry. By means of pharmacological inhibitors, dominant negative mutants, and siRNA of various cellular proteins and signaling molecules, phosphorylation of caveolin-1, dynamin-2 expression, and Rac1 activation were suppressed, suggesting that by orchestrating p38 MAPK, Src, and Rac1 signaling cascade in the target cells, ARV creates an appropriate intracellular environment facilitating virus entry and productive infection. Furthermore, disruption of microtubules, Rab5, or endosome acidification all inhibited ARV infection, suggesting that microtubules and small GTPase Rab5, which regulate transport to early endosome, are crucial for survival of ARV and that exposure of the virus to acidic pH is required for productive infection.
authors.affiliation: Institute of Molecular Biology, National Chung Ching University, Taichung 402.
authors.forename: Wei R
authors.initials: WR
authors.lastname: Huang
authors.affiliation: Institute of Molecular Biology, National Chung Ching University, Taichung 402; Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 912.
authors.forename: Ying C
authors.initials: YC
authors.lastname: Wang
authors.affiliation: Institute of Molecular Biology, National Chung Ching University, Taichung 402; Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 912.
authors.forename: Pei I
authors.initials: PI
authors.lastname: Chi
authors.affiliation: Institute of Molecular Biology, National Chung Ching University, Taichung 402.
authors.forename: Lai
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Veterinary Medicine, National Chung Ching University, Taichung 402.
authors.forename: Chi Y
authors.initials: CY
authors.lastname: Wang
authors.affiliation: Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 11221, Taiwan.
authors.forename: Chi H
authors.initials: CH
authors.lastname: Lin
authors.affiliation: Institute of Molecular Biology, National Chung Ching University, Taichung 402. Electronic address: hjliu5257@nchu.edu.tw.
authors.forename: Hung J
authors.initials: HJ
authors.lastname: Liu
mesh_terms: D000818:Animals; D051242:Caveolin 1 / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism; D034285:Dynamin II / Q000378:metabolism*; D004705:Endocytosis; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry*; D030101:Orthoreovirus, Avian / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D014709:Vero Cells; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D034741:RNA, Small Interfering; D002784:Cholesterol; D019061:src-Family Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020559:Monomeric GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D034285:Dynamin II