S7value was calculated using a two-tailed Students test. KSHV-infected LR fractions with anti-31 antibodies and analyzed them by mass spectrometry. YK 4-279 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 (31 and V3) in LRs early during YK 4-279 contamination. Preincubation of computer 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, computer virus internalization, and productive nuclear trafficking of KSHV DNA. Taken together, these studies demonstrate that this EphA2 receptor acts as a grasp 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 contamination. and and show the enlarged images of the boxed areas. (Scale bar: 10 m.) (and and and and 0.00016). There was no change in the levels of integrins 1 and 3 after 10 min p.i., further confirming that KSHV induced a rearrangement of integrins at the cell surface LRs without increasing their total protein levels (Fig. S2). In contrast, EphrinB2 (EphB2), a class-B Ephrin receptor, did not show any increased colocalization with integrin 31 in infected cells compared with uninfected cells (Fig. S3), which demonstrated the specificity of KSHV interactions with EphA2. At 10 min p.i., we also observed the colocalization of EphA2 with KSHV as detected by envelope glycoprotein gpK8.1A (Fig. 2and is usually depicted in the 0.00016). (as the enlarged image. (Scale bar: 10 m.) EphA2 shRNA Rabbit Polyclonal to ADCY8 Inhibits KSHV Entry and Contamination of HMVEC-d Cells. To characterize the functional effect of EphA2 on KSHV contamination, we tested five different EphA2 lentivirus-encoding shRNAs to determine the knockdown of EphA2 levels in HMVEC-d cells (Fig. 3and = 0.0001). (0.005). (and Fig. S4and immunostained for EphA2, p-myosin IIA, and LR marker (Flotillin-1). Representative images are shown. The boxed region in the merged panel is usually enlarged and shown in the and and and Fig. S6 and and and Fig. S7value was calculated using a two-tailed Students test. (and Fig. S7and Fig. S70.0004). Line-scan analysis of the enlarged cells clearly revealed the synchronized red signals (KSHV) within the green signal peaks (Rab5) in control shRNA cells but lacking in EphA2 shRNA-transduced cells (Fig. 6and for details. Antibodies and Reagents. See for details. Mass Spectrometry. Serum-starved (8 h) HMVEC-d cells were either mock or KSHV (10 DNA copies per cell) infected for 5 min. LR and nonlipid raft (non-LR) fractions were isolated. Two hundred micrograms protein from LR and non-LR fractions was immunoprecipitated with mouse anti-31 and control antibody. Immunoprecipitates were separated by 10% SDS/PAGE and the bands specific for 31 immunoprecipitates were analyzed by mass spectrometry, using LC-electrospray ionization (ESI)-MS methods at the Midwest Proteome Center, Rosalind Franklin University of Medicine and Sciences. Measurement of KSHV Binding, Entry, and Nuclear Delivery by Real-Time DNA PCR. HMVEC-d cells were infected with KSHV (10 DNA copies per cell) at 4 C (binding) or 37 C YK 4-279 (entry and nuclear delivery) for 1 h. Details are provided in for details. Measurement of KSHV Contamination in the Presence of Soluble EphA2. Ten DNA copies per cell of KSHV were preincubated with 10 g/ml recombinant soluble EphA2 (Sol-EphA2) (R&D Systems) for 1 h at 37 C. HMVEC-d cells were infected with unincubated or Sol-EphA2 incubated KSHV for 1 h at 37 C. KSHV DNA binding, entry, and gene expression were measured as described in for details. Quantification of Macropinocytic Blebs. Determination of blebs in KSHV-infected cells was performed as described previously (11) ( em SI Materials and Methods YK 4-279 /em ). Supplementary Material Supporting Information: Click here to view. Acknowledgments We thank Dr. Horonori Katoh for the nice gift of myc-tagged EphA2 plasmid. We also thank Keith Philibert and Dr. Alice Gilman-Sachs for critically reading the manuscript and Dr. Xinli Yang, Midwest Proteome Center [National Institutes of Health Grant National Center for Research Resources (NCRR) S10RR19325], Rosalind Franklin University of Medicine and Science, for mass spectrometry. This study was supported in part.