Assembly and characterization of EBV-gp350/220-F VLPs. (A) Schematic diagram showing cDNAs pCAGGS-NDV-M, −NDV-NP and -EBVgp350/220-F co-transfected into cells followed by VLP assembly and release. (B) Supernatants containing independent EBV-VLP preparations were harvested daily between 24–96 h, concentrated and purified by sucrose-gradient centrifugation followed by particle lysis and immunoblot. mAb-72A1 anti-gp350/220 (left two panels), polyclonal anti-HR2 (third panel) and polyclonal anti-NDV (right panel) detected EBVgp350/220 ED and NDV-F C-terminal peptides respectively. Anti-NDV also detected M and NP in VLPs and NDV, but not EBV. Lanes 1–3 contain VLPs derived from 293T, lanes 6,7,10,11,14, 15 from CHO cells. Lysates of purified EBV (lanes 4, 8, 12, 16) and NDV (lanes 5, 9, 13) were controls. (C) Different combinations of pCAGGS plasmids encoding F, M, NP, gp350/220 and gp350/220-F as indicated were co-transfected into 293T cells. Supernatant content was pelleted and analyzed by immunoblot using anti-gp350/220 and anti-HR2 antibodies. Lane 1: NDV-VLP (NP, M, F), lane 2: NP, M, lane 3: NP, M, gp350/220 WT, lane 4: EBV-VLP (NP, M, EBVgp350/220-F), lane 5: M, EBVgp350/220-F, lane 6: pCAGGS . Both antibodies in lane 4 alone detected bands of the expected MW, indicating a chimeric EBV-VLP was specifically assembled and released. (D) Electron micrograph of negatively stained sucrose gradient purified EBVgp350/220-F VLPs prepared in CHO cells compared with native EBV -/+ immunogold-coupled antibodies. Top, structure and size of the chimeric VLP compared with EBV without antibody. Middle, demonstration that mAb-72A1 detected by immunogold-coupled goat anti-mouse IgG binds the surface of a chimeric EBVgp350/220-F VLP and EBV (control). Bottom, but not a chimeric KSHV-VLP or KSHV. (E) Silver stain of increasing amounts of purified chimeric VLPs released from ELL-0 cells compared with NDV. Positions of EBVgp350/220-F, NDV-NP and -M are indicated by arrows. MW markers are at left.