Figure 1
Transiently expressed HCF-1 protein promotes the synthesis of NPV DNA and proteins in non-permissive Tn368 cells. Tn368 cells were transfected with 2 μg of pFBD/luc (Luc) or pFBD/hcf-1 (HCF-1) DNA, which express luciferase or HCF-1 protein, respectively, under control of the heat shock protein 70 promoter (hsp70 pro). At 24 h post-transfection, the transfected cells were heat-shocked at 42 °C for 30 min, incubated for 6 h at 28 °C, and were then infected with HycuMNPV (Hycu), OpMNPV (Op), BmNPV (Bm) and LdMNPV (Ld) at MOIs of 10, 50, 10 and 3, respectively. (a) Schematic representation of the plasmids pFBD/luc and pFBD/hcf-1 used in these experiments. (b) Microscopic images of Tn368 cells at 72 h post-infection. The arrowheads indicate polyhedra-containing cells. Scale bar, 50 μm. (c) BV yields in virus-infected Tn368 cells at 72 h post-infection (pi). BV titers were determined by a plaque assay using SpIm cells for HycuMNPV, BM-N cells for BmNPV and Ld652Y cells for OpMNPV and LdMNPV. The vertical bars represent the standard deviations of the averages from three determinations. (d) Viral DNA production at 24 h post-infection. Viral DNA was quantified by qPCR. The vertical bars represent the standard deviations of the averages from three determinations. (e) Immunoblot analysis of VP39 and polyhedrin (Polh) proteins at 72 h post-infection. Polypeptides from infected Tn368 cells were resolved on 12% SDS-polyacrylamide gels and transferred onto Immobilon-P (for VP39 protein) or nitrocellulose membranes (for polyhedrin). VP39 protein was probed using anti-BmNPV/AcMNPV VP39 polyclonal antibody and visualized with ECL Select Western Blotting Detection Reagent. Polyhedrin protein was probed with anti-BmNPV polyhedrin polyclonal antibody and visualized using a HRP Conjugate Substrate Kit. (f) Immunoblot analysis of luciferase and HCF-1 proteins in infected Tn368 cells. Luciferase and HCF-1 proteins were probed by anti-HA monoclonal antibody and visualized using ECL Western Blotting Detection Reagent and ECL Select Western Blotting Detection Reagent, respectively. In panels (e) and (f), polypeptides equivalent to 1 × 105 cells were analyzed in each lane.
