P40 subcellular distribution in AcMNPV-infected cells.

(A). Subcellular distribution of P40 in plasmid-transfected cells. P40-V₅ was transiently expressed in Sf9 cells by plasmid transfection and then the cells were either mock-infected or infected with vAc^egfp (MOI = 2). At 24 hpi, the cells were subjected to either immunofluorescence microscopy (left panel) or cell fractionation (right panel). For immunofluorescence microscopy, the cells were fixed, probed with anti-V₅ antibody to detect P40-V₅, and visualized with Alexa Fluor 568-conjugated secondary antibody (red). Hoechst 33258 was used to stain the nuclear DNA (blue). For the cell fractionation assay, the cells were harvested and separated into nuclear (N) and cytoplasmic (C) fractions. The fractions were separated by SDS-PAGE and probed with anti-V₅, anti-tubulin, and anti-histone-H₃ antibodies to detect P40, tubulin, and histone-H₃, respectively. (B). EGFP-P40 subcellular distribution. EGFP-P40 was expressed in mock-infected Sf9 cells or cells infected with vAc^polh (MOI = 2), a bacmid derived from the AcMNPV genome with an inserted polyhedrin open reading frame (ORF). At 48 hpt, the cells were analyzed by fluorescence microscopy. (C). EGFP-P40 associates with Arp2 and P20. EGFP (approx. 27 kDa) or EGFP-P40 (approx. 69 kDa) was co-expressed with Arp2-Ha (approx. 46 kDa) or P20-Ha (approx. 21 kDa) in Sf9 cells, respectively. At 48 hpt, the cells were harvested and subjected to a co-IP assay using anti-Ha. The immunoprecipitated proteins and the WCL were separated by SDS-PAGE and probed with anti-Ha and anti-EGFP. (D). AcMNPV late gene products induce P40 nuclear accumulation in the late infection phase. Sf9 cells transfected with the P40-V₅ encoding plasmid were either mock infected or infected with vAc^egfp (MOI = 2) in the presence or absence of APH. The cells were fixed at 6, 12 and 24 hpi or harvested for fractionation and further treated as described in (A). Scale bar: 20 μm; Scale bar in the zoomed panel: 10 μm.