Kaposi's sarcoma-associated herpesvirus (KSHV), etiologically associated with
Kaposi's sarcoma, uses
integrins (α3β1, αVβ3, and αVβ5) and associated signaling to enter human dermal microvascular endothelial cells (HMVEC-d), an in vivo target of
infection. KSHV
infection activated c-Cbl, which induced the selective translocation of KSHV into
lipid rafts (LRs) along with the α3β1, αVβ3, and xCT receptors, but not αVβ5. LR-translocated receptors were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVβ5 was polyubiquitinated, leading to
clathrin-mediated entry that was targeted to lysosomes. Because the molecule(s) that integrate signal pathways and productive KSHV macropinocytosis were unknown, we immunoprecipitated KSHV-infected LR fractions with anti-α3β1
antibodies and analyzed them by mass spectrometry. The
tyrosine kinase EphrinA2 (EphA2), implicated in many
cancers, was identified in this analysis. EphA2 was activated by KSHV. EphA2 was also associated with KSHV and
integrins (α3β1 and αVβ3) in LRs early during
infection. Preincubation of virus with soluble EphA2, knockdown of EphA2 by shRNAs, or pretreatment of cells with anti-EphA2
monoclonal antibodies or
tyrosine kinase inhibitor dasatinib significantly reduced KSHV entry and gene expression. EphA2 associates with c-Cbl-
myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to
bleb formation and macropinocytosis of KSHV. EphA2
shRNA ablated macropinocytosis-associated signaling events, virus internalization, and productive nuclear trafficking of KSHV
DNA. Taken together, these studies demonstrate that the
EphA2 receptor acts as a master assembly regulator of KSHV-induced signal molecules and KSHV entry in endothelial cells and suggest that the
EphA2 receptor is an attractive target for controlling KSHV
infection.