Editors' ChoiceMicrobiology

A Gatekeeper of Viral Entry

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Sci. Signal.  22 May 2012:
Vol. 5, Issue 225, pp. ec147
DOI: 10.1126/scisignal.2003246

Kaposi’s sarcoma–associated herpesvirus (KSHV) can enter cells through macropinocytosis, hijacking host signaling pathways to do so. In human dermal microvascular endothelial (HMVEC-d) cells, KSHV interacts with integrins, resulting in the induction of focal adhesion kinase (FAK), the tyrosine kinase Src, phosphoinositide 3-kinase (PI3K), guanosine triphosphatases (GTPases) of the Rho family, and other signaling molecules. KSHV entry requires translocation to lipid rafts, which involves the E3 ubiquitin ligase c-Cbl. Chakraborty et al. immunoprecipitated KSHV-infected lipid raft fractions from HMVEC-d cells with antibodies to α3β1 integrin and performed mass spectrometry on the immunoprecipitated proteins. They identified the receptor tyrosine kinase EphrinA2 (EphA2) as a molecule associated with integrins in KSHV-infected cells. Ephrins and their receptors modulate endothelial cell migration and vascular assembly during angiogenesis and affect the actin cytoskeleton, cell-substrate adhesion, and intercellular junctions. Immunofluorescence microscopy showed that phosphorylated EphA2 colocalized with the lipid raft marker flotilin-1 in KSHV-infected cells and that EphA2 colocalized with integrins at the edges of infected cells. Knockdown of EphrA2 with short hairpin RNA (shRNA) reduced the amount of KSHV viral DNA in cells and inhibited expression of KSHV latent genes. Treatment with antibodies that blocked EphA2 or the EphA2 inhibitor dasatinib before infection also reduced the amount of viral DNA in the cells. EphA2 coimmunoprecipitated with c-Cbl in infected HMVEC-d cells and colocalized with both c-Cbl and α3β1 integrin as shown by triple immunofluorescence. In infected cells, EphA2 coimmunoprecipitated and colocalized with both Src and PI3K. Phosphorylated Src, PI3K, and c-Cbl were abundant in lipid raft fractions of KSHV-infected cells, and their phosphorylation was reduced in lipid raft fractions from infected cells in which EphA2 was knocked down. Phosphorylated EphA2 localized in macropinocytic structures and knockdown of EphA2 reduced the number of these structures. Knockdown of EphA2 also inhibited endocytic uptake of KSHV, as shown by tracking DiI-labeled KSHV and the macropinocytic marker dextran. Thus, EphA2 appears to enhance KSHV entry by promoting signals that trigger macropinocytosis.

S. Chakraborty, M. V. Veettil, V. Bottero, B. Chandran, Kaposi’s sarcoma-associated herpesvirus interacts with EphrinA2 receptor to amplify signaling essential for productive infection. Proc. Natl. Acad. Sci. U.S.A. 109, E1163–E1172 (2012). [Abstract] [Full Text]

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