The Eph RTK interaction maps to the N-terminal region of KSHV gH.

A Co-Immunoprecipitation of depicted V5-tagged RRV/KSHV gH chimeras in complex with KSHV gL-Flag identifies KSHV gH domains necessary for binding of EphA2. Monoclonal antibody to the V5-tag was used for precipitation of complexes. Equal amounts of HA epitope-tagged EphA2 ectodomain was added to each reaction. EphA2, gH, gL were detected using their respective tag. RRV-derived parts of the chimeric constructs are displayed in light grey; lane 1: KSHV gH, lane 9: RRV gH. Four gH chimeras (lanes 3 and 5–7) were not expressed to detectable levels. All chimeras harboring domain I of KSHV gH (lanes 2, 4, 8) were still able to interact with EphA2. B Co-Immunoprecipitation of V5-tagged KSHV gH in complex with Flag-tagged RRV/KSHV gL chimeras identifies KSHV gL domains necessary for binding of EphA2. gH-V5/gL-Flag complexes were immunoprecipitated in the presence of full-length EphA2-myc using monoclonal antibody to the V5-tag and precipitates were analyzed by Western blot as in A. RRV-derived parts of the chimeric constructs are displayed in light grey; first lane: KSHV gL, fourth lane: RRV gL. C Co-Immunoprecipitation of V5-tagged KSHV gH in complex with a Flag-tagged C-terminally truncated KSHV gL mutant (gLΔ135–164). gH-V5/gL-Flag complexes were immunoprecipitated in the presence of EphA2-HA (ectodomain) using monoclonal antibody to the V5-tag and precipitates were analyzed by Western blot as in A. Full-length KSHV gH/gL serves as a positive control, KSHV gH alone serves as a negative control. Asterisks indicate non-specific bands. Abbreviations: D: domain, TM: transmembrane domain, SP: signal peptide, IVD: intravirion domain, IP: immunoprecipitation, IB: immunoblotting.