EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

Dipanjan Dutta,Sayan Chakraborty,Chirosree Bandyopadhyay,Mohanan Valiya Veettil,Mairaj Ahmed Ansari,Vivek Vikram Singh,Bala Chandran,Blossom Damania

doi:10.1371/journal.ppat.1003510

Dipanjan Dutta, Sayan Chakraborty + Show 6 more

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https://doi.org/10.1371/journal.ppat.1003510

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Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2's tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection.

Highlights

During the initiation of infection of target cells, viruses bind to the cellular receptors and utilize a plethora of cellular signal molecules
To initiate its in vitro infection of endothelial cells, Kaposi’s sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate, integrins, and EphrinA2 (EphA2) molecules in the lipid raft (LR) regions, which induces the integrin-c-Cbl associated signaling and macropinocytic entry
The present studies conducted to define the key molecules regulating KSHV entry in human foreskin fibroblast (HFF) cells demonstrate that KSHV induces the association of integrins with EphA2 in the non-LR regions of HFF cells and activates EphA2, which in turn associates with c-Cbl, myosin IIA, FAK, Src, PI3-K, clathrin, AP2 and Epsin15

Summary

Introduction

During the initiation of infection of target cells, viruses bind to the cellular receptors and utilize a plethora of cellular signal molecules. Besides fusion of the viral envelope with the host plasma membrane, receptor mediated endocytosis, an essential biological process mediating cellular internalization events, is often exploited by many enveloped and non-enveloped viruses for their entry into target cells [2,3]. KSHV, etiologically associated with Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multi-centric Castleman’s disease (MCD), manifests a wide range of receptor(s) and signal molecules utilization that varies according to the target cell type, serving as an excellent model to determine virus entry associated events [4,5,6]. Following de novo infection of skin-derived fibroblasts, KSHV induces the production of pro-inflammatory and pro-migratory factors and promotes endothelial cell invasion of extra cellular matrix (ECM) through paracrine mechanisms [9]. Latent KSHV infection of oral cavity derived primary human fibroblasts enhances the secretion of KS-promoting cytokines and intrinsic invasiveness through VEGF-dependent mechanisms [10], which highlight the potential role for KSHV-infected fibroblasts in promoting KS pathogenesis

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