FIG. 4.

Replication of five different NPVs in HRF-1-expressing Ld652Y cells. (A) Schematic representation of the pGL3-Basic vector (Promega)-based construct used for transfection. pHyHr6IE1/HA-HRF1 (a) and pHyHr6Ie1/EGFP (b) contained the HA-fused hrf-1 gene and the egfp gene, respectively, both regulated by the hycu-ie1 promoter enhanced by hycu-hr6. B, BamHI; E, EcoRI; N, NcoI; X, XbaI. (B) Monolayer cultures of Ld652Y cells (10⁶ cells) were transfected with 1 μg of pHyHr6IE1/HA-HRF1 (HRF-1) or pHyHr6IE1/EGFP (EGFP). At 24 h posttransfection, transfected cells were infected with AcMNPV (Ac), BmNPV (Bm), SeMNPV (Se), HycuNPV (Hycu), and SpltMNPV (Splt) at an MOI of 1 PFU/cell and were examined for cytopathology at 96 hpi. Arrowheads indicate the cells containing polyhedra. (C) Accumulation of HA-fused HRF-1 and polyhedrin in transfected and infected cells were monitored by immunoblotting with antisera against HA (HA-HRF-1; upper panel) and BmNPV polyhedrin (Polh; lower panel). The numbers on the left indicate the molecular masses (kilodaltons) of marker proteins. (D) Ld652Y cells were transfected with genomes of five different NPVs together with either pHyHr6IE1/HA-HRF1 (HRF-1) or pHyHr6IE1/EGFP (EGFP). BV yields in culture medium from transfected cells at 96 h posttransfection were determined by plaque assay using cell lines permissive for each NPV: Sf9, BmN4, Se301, SpIm, and SpLi cells for AcMNPV, BmNPV, SeMNPV, HycuNPV, and SpltMNPV, respectively. Vertical bars indicate the standard deviations from three determinations. ND, less than 1 PFU/ml.