FIG. 7.

Transient DNA replication assays and expression of P143. (A) Sf21 cells were transfected with a collection of plasmids which together expressed AcMNPV genes necessary for plasmid DNA replication (ie1, dnapol, lef1, lef2, p35, pe38, and ie2) plus p143 and lef-3 (lane 1), only p143 (lane 2; no LEF-3), only lef-3 (lane 3; no P143), lef-3 plus lef3(2-56)p143 (lane 4) or only lef-3(2-56)p143 (lane 5; no LEF-3). All of these constructs expressed proteins under their endogenous promoters. Following incubation for 48 h, total intracellular DNA was prepared and digested with HindIII to linearize the plasmids, and DpnI to digest nonreplicated plasmid DNA. Southern blots of these restriction-digested DNA preparations were probed with a digoxigenin-labeled PCR product of the AcMNPV ie-1 gene. (B) Sf21 cells were transfected with pAcP143 (P143) or pAcLEF3(1-56)-P143 (1-56-P143). At 24 h posttransfection, the cells were prepared for immunofluorescence microscopy and probed with monoclonal anti-AcMNPV P143 antibody. Immunofluorescence was detected using a secondary goat anti-mouse immunoglobulin G conjugated with Alexa Fluor 568. Nuclei were detected by DAPI staining. The red immunofluorescence P143 stain is superimposed with the blue DAPI nuclear stain. (C) Similarly transfected cells were harvested at 48 h posttransfection, and biochemically fractionated into cytoplasmic (C) and nuclear (N) extracts that were analyzed by immunoblotting and probing with anti-P143 antibody. Whole-cell extracts (W) were included as controls.