Baculovirus infection raises the level of TATA-binding protein that colocalizes with viral DNA replication sites

Abstract

During the infection cycle of Autographa californica multicapsid nuclear polyhedrosis virus, the TATA-binding protein (TBP) of the insect host cell likely participates in early viral transcription, which is mediated by the host RNA polymerase II. However, the role of TBP in late and very late viral transcription, which is accomplished by an alpha-amanitin-resistant RNA polymerase, is unclear. We observed a dramatic increase of TBP protein during the late phases of infection. TBP mRNA levels, however, were not coordinately increased. Indirect-immunofluorescence studies revealed a nuclear redistribution of TBP during infection. After labeling of viral replication centers with bromodeoxyuridine (BrdU), costaining of TBP and BrdU showed that TBP localized to viral DNA replication centers. These results suggest a putative role of TBP during late viral transcription, which may occur in close proximity to viral DNA replication.

FIG. 1.

Expression of TBP in AcMNPV-infected TN-368 and S. frugiperda cells. (A) Detergent-based nuclear extracts were prepared from uninfected S. frugiperda (lane 0, nuc S. frugiperda) or TN-368 cells (lane 0, nuc TN-368) or from cells infected at 2, 4, 8, 16, 24, 48, and 72 h p.i. Proteins were resolved on sodium dodecyl sulfate-10% polyacrylamide gels and stained with the polyclonal anti-Sf/TBP antiserum raised in rabbits. As control, the Western blot with the protein extracts of TN-368 cells was also stained with the anti-IE2 antiserum (11). The arrowheads on the right indicate the TBP protein of about 36 kDa and the IE2 protein of 49 kDa. Protein size markers are given on the left. (B) Total RNA (100 μg) isolated from uninfected S. frugiperda cells (lane 0) or from cells at 4, 8, 16, 24, 48, and 72 h p.i. (lanes 4, 8, 16, 24, 48, and 72) was analyzed on an 1.2% agarose gel containing 2.2 M formaldehyde. The Northern blot was hybridized to a TBP-specific RNA probe of 369 nucleotides. The schematic representation of the TBP open reading frame, with the putative transcriptional start site, and the position of the RNA probe are given below. The box indicates the T7 promoter.

FIG. 2.

Specificity of the TBP protein in insect cells. Detergent-based nuclear extracts were prepared from uninfected S. frugiperda (lane 0) or from cells infected at 2, 8, 16, 24, and 48 h p.i. Proteins were resolved on sodium dodecyl sulfate-10% polyacrylamide gels and stained with rabbit anti-TBP antiserum directed against the N terminus of Sf/TBP or with MAb 58C9 directed against the C terminus of Drosophila TBP. The arrowheads indicate the TBP protein band, and protein size markers are given on the left.

FIG. 3.

Localization of TBP and colocalization with viral DNA replication sites after AcMNPV infection of S. frugiperda cells. (A) Uninfected and AcMNPV-infected S. frugiperda cells were fixed in 2% paraformaldehyde and permeabilized in 0.1% Triton X-100 at 4, 8, 16, 24, and 48 h p.i. Staining was performed with the anti-Sf/TBP antiserum and fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulin (Jackson Laboratory) (a to g). The specimens were viewed using a Zeiss Axiovert 135 microscope linked to the INTAS digital camera system (a to i). As a control, cells at 4 h p.i. were stained with rabbit preimmune serum (a). To visualize DNA replication, uninfected S. frugiperda cells and cells at 8 h p.i. were stained first with BrdU and then with mouse MAb BrdU (red) as previously described (13) (h and i). (B) Cells at 8 h p.i. were costained with rabbit anti-Sf/TBP antiserum (green) and mouse MAb BrdU (red). For confocal imaging, a Leica DM IRBE microscope linked to Leica TCS-SP was used. Confocal images and the merged image are shown.