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

Wei R Huang,Ying C Wang,Pei I Chi,Lai Wang,Chi Y Wang,Chi H Lin,Hung J Liu

doi:10.1074/jbc.m111.257154

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.

Highlights

IntroductionNSC99-2321-B-005-015-MY3 and NSC97-2313-B-005-048-MY3. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. 1 To whom correspondence should be addressed
NSC99-2321-B-005-015-MY3 and NSC97-2313-B-005-048-MY3. □S The on-line version of this article contains supplemental Figs. 1 and 2. 1 To whom correspondence should be addressed
Using specific inhibitors and hypertonic medium, together with siRNAs, DN mutants, and Csk, we demonstrate that avian reovirus (ARV) enters host cells through the caveolin-1-mediated and dynamin-2-dependent pathways and that ARV-induced activation of Ras, p38 MAPK, and Src facilitates virus entry and activation of Rac1 that is beneficial for virus replication

Summary

Introduction

NSC99-2321-B-005-015-MY3 and NSC97-2313-B-005-048-MY3. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. 1 To whom correspondence should be addressed. In clathrin-mediated endocytosis, clathrin is first recruited to the plasma membrane in response to receptor-mediated internalization signals, which results in the assembly of CCPs2 at the cytoplasmic side of the cell membrane. A number of viruses enter into host cells through the clathrin-dependent endocytosis pathway [3,4,5]. Internalization of SV40 was found in cells that do not express caveolin-1, suggesting that SV40 utilizes the caveoladependent pathway and the lipid raft-dependent and caveola-independent pathway [18]. The binding event either triggers membrane fusion at the plasma membrane or internalization of the virus into an endosome. The avian reovirus fiber is formed by capsid protein ␴C and is encoded by the third open reading frame of the S1 segment, which is responsible for host cell attachment [25, 26] and induction of apoptosis [27]

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