FIG. 2.

Immune system activation by AcNPV in macrophages is not mediated by gp64. (A) Wild-type gp64 and a deletion mutant lacking the transmembrane region of the gp64 envelope protein (gp64ΔTM) were expressed in Sf-9 cells. Whole-cell lysates and culture supernatants were subjected to SDS-PAGE under reducing conditions and visualized by immunoblotting with an antihexahistidine monoclonal antibody. Lane 1, cells transfected with pIB/V5-His; lanes 2 and 3, cells transfected with pIBgp64ΔTM/V5-His and pIBgp64/V5-His, respectively. The heavy chains of the antibody are indicated by asterisks. (B) Purified AcNPV virions (lane 2) and gp64ΔTM (lane 3) were analyzed by SDS-PAGE and Coomassie blue staining. Lane 1, molecular mass markers. (C) Activation of mouse macrophage RAW264.7 cells (10⁶ cells/well) treated with the indicated amounts of AcNPV or gp64ΔTM. The production of TNF-α and IL-6 in culture supernatants after 24 h of incubation was determined by sandwich ELISAs. PGN was used as a positive control. Data are shown as means ± SD. (D) Production of IFN-α in RAW264.7 cells (10⁶ cells/well) inoculated with AcNPV (5 μg/ml) or gp64ΔTM (5 μg/ml), as determined by a sandwich ELISA after 24 h of incubation. Data are shown as means ± SD. (E) Production of TNF-α in RAW264.7 cells (10⁶ cells/well) inoculated with AcNPV (20 μg/ml) or PGN (2.5 μg/ml), with or without a pretreatment with the indicated amounts of gp64ΔTM for 2 h at 37°C. After 24 h of incubation, the production of TNF-α in culture supernatants was determined by a sandwich ELISA. Data are shown as means ± SD.