ERK MAP kinase-activated Arf6 trafficking directs coxsackievirus type B3 into an unproductive compartment during virus host-cell entry

Abstract

Clathrin- and caveolae-mediated endocytosis have been implicated in the productive entry of many viruses into host cells. ADP-ribosylation factor 6 (Arf6)-dependent endocytosis is another endocytosis pathway that traffics from the cell surface and it is the only Arf that traffics at the plasma membrane. However, little is known about Arf6-dependent trafficking during virus entry. This study showed that coxsackievirus type B3 (CVB3) associated with decay-accelerating factor in non-polarized HeLa cells can be redirected into non-productive compartments by Arf6-dependent internalization, thus restricting infection. Overexpression of wild-type (WT) and constitutively active (CA) Arf6 in HeLa cells resulted in a 2.3- and 3.6-fold decrease in infection, respectively. A dominant-negative inhibitor of Arf6 recovered restriction of infection by WT-Arf6 and CA-Arf6. RNA interference of endogenous Arf6 resulted in a 3.3-fold increase in CVB3 titre in HeLa cells. It was shown that coxsackie–adenovirus receptor (CAR) ligation by virus or CAR-specific antibody could activate extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase family and lead to Arf6-mediated viral restriction. In the absence of ERK activation, CVB3 internalization into early endosomes was inhibited and subsequent infection was reduced, but Arf6-mediated restriction was also abolished. In conclusion, receptor-mediated signalling enhances CVB3 entry whilst also activating non-productive pathways of virus entry; thus, virus infection is an equilibrium of productive and non-productive pathways of entry.

Dynamin- and Lipid Raft-Dependent Entry of Decay-Accelerating Factor (DAF)-Binding and Non-DAF-Binding Coxsackieviruses into Nonpolarized Cells

“…Our observations are consistent with reports by other investigators that dynamin is required for CVB3 infection (13,33).…”

“…Other investigators have provided evidence that in HeLa cells CVB3 infection involves dynamin (13,33), as well as clathrin and endosomal acidification (13). However, the reagents used to demonstrate the roles of clathrin and acidification were not highly specific, endocytic inhibitors were not shown specifically to affect virus entry rather than infection, and it was not made clear whether the CVB3 isolate studied did or did not bind to DAF.…”

WNT3A induces a contractile and secretory phenotype in cultured vascular smooth muscle cells that is associated with increased gap junction communication

“…Cells were coverslipped with VectaShield mounting medium containing DAPI (Vector Laboratories) and images were captured using a Leica AOBS SP2 confocal microscope as we have described previously. 21,22 Antibodies used for staining were as follows: b-catenin (BD Biosciences, product number 610154) and connexin 43 (Cell Signaling, product number 3512).…”

Cellular and Immunological Regulation of Viral Myocarditis

“…The activation of ERK MAP kinase was first reported in the context of CVB3 infection both in vitro (Luo et al, 2002) and in vivo (Opavsky et al, 2002). The activation of this molecule by virus binding the CAR receptor may be involved in triggering endocytosis uptake of CVB3 during entry (Marchant et al, 2009b). The activation of p38 MAP kinase was later shown to be required for cell lysis and release of progeny virion.…”