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First published online June 8, 2006
doi: 10.1242/10.1242/jcs.02982

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Nucleolar protein upstream binding factor is sequestered into adenovirus DNA replication centres during infection without affecting RNA polymerase I location or ablating rRNA synthesis

¹ Division of Virology, Department of Cellular and Molecular Medicine, University Walk, University of Bristol, Bristol, BS8 1TD, UK
² Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK

View larger version (47K): [in a new window]   Fig. 1. UBF is localised next to viral replication centres. (A-C) UBF is shown in green (anti-UBF serum), DBP in red and the location of the nucleoli is revealed by phase contrast. (A) The location of UBF in uninfected HeLa cells relative to the nucleoli. (B) The location of UBF in adenovirus-infected cells at 16 hours post-infection is shown relative to viral DBP and the nucleoli. (C) Adenovirus-infected cells were examined at 8 hours post-infection when the replication centres are just beginning to form. (D) The location of preTP (in red) and UBF (in green) was examined in infected cells at 18 hours post-infection. The inset in the final merged image is a close up to illustrate that UBF and preTP staining is proximal but does not overlap in the main. All images are of a single focal plane approximately 0.3 µm depth, bar, 10 µm. (E) A western blot of equal numbers of either uninfected cells or adenovirus-infected cells after 16 hours, probed with anti-UBF antiserum to reveal total quantities of protein. The location of the molecular mass markers is shown on the left.

View larger version (47K): [in a new window]   Fig. 2. The location of different mutations of EGFP-UBF in uninfected and infected cells. (A) A schematic diagram of UBF. The top drawing indicates the relative location of the 6 HMG boxes (numbered 1-6), the dimerisation domain (cross hatching) and the transactivation domain (marked with dots). The six schematics below indicate the position of EGFP (shaded box), the relative extent of the UBF deletion mutant fused to the C-terminus of EGFP and the exact number of amino acids from UBF fused to EGFP. (B) The usual location of each fusion protein in HeLa cells at 18 hours post-transfection, B23 is in red and the EGFP fusion is in green. (C) The cells were infected with adenovirus and transfected with plasmid encoding the appropriate fusion protein. The EGFP fusion is shown in green and the location of viral DBP is shown in red. All images are of a single focal plane approximately 0.3 µm depth; bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 3. Location of viral nucleic acids and nucleic acid synthesis relative to UBF in adenovirus-infected cells. (A) A fluorescence in situ hybridisation analysis shows a cell 18 hours post-infection with adenovirus adjacent to an uninfected cell which serves as a control for the probe. The location of UBF is shown in green, the DIG-labelled adenovirus DNA is shown in red and the nucleus is revealed by DAPI staining, and is in blue. (B) At 18 hours post-infection, cells were treated with FU to reveal sites of RNA synthesis. UBF is in green, the newly synthesised RNA is in red and viral DBP is in blue. (C) The cells were labelled at 18 hours post-infection with BrdU followed by a chase period of 2 hours. UBF is in green and BrdU is in red. As stated in the Materials and Methods the cells were pretreated with HCl to reveal the location of all the incorporated label, both ssDNA and dsDNA. (D) The pulse period was short, there was no chase and the HCl treatment was omitted. Again, UBF is in red, BrdU is in green and DAPI is in blue. All images are of a single focal plane approximately 0.3 µm depth; bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 4. ChIP assay reveals UBF preferentially precipitates sequences near the ends of the viral genome. Three different samples of target DNA were used to amplify different regions of the viral genome as indicated along the top. Either purified Ad2 DNA, or virally infected cells subjected to ChIP using anti UBF serum or virally infected cells subjected to ChIP using the matched pre-immune sera. In each case four different primer pairs were used to amplify, by PCR, four different regions of viral DNA, viz. sigma (250 bp from left hand end), E1 promoter (450 bp from left hand end), Major late promoter (6000 bp from left hand end), VA RNA I (12000 bp from left hand end), Fibre gene (4000 bp from right hand end) and E4 ORF3 (1500 bp from right hand end). The PCR products were separated on agarose gels and photographed as shown. The primer pair used in each sample is indicated at the top of the gel. On the right hand side the size of two markers are indicated, the intervening marker sizes are 400 bp, 350 bp and 300 bp.

View larger version (60K): [in a new window]   Fig. 5. Double strand break repair (DSBR) protein, Rad50, is associated with UBF in cells infected with E4 deleted virus, dl366. HeLa cells were infected or mock-infected with the mutant virus and fixed at 18 hours post-infection. (A) An infected cell showing the location of Rad50 (in red), which is seen alongside DBP (in green). In the merged image, DAPI is included to illustrate the extent of the nucleus. (B) Two uninfected cells, with Rad50 in red and UBF in green. (C) An infected cell, labelled as in B. All images are of a single focal plane approximately 0.3 µm depth; bar, 10 µm.

View larger version (84K): [in a new window]   Fig. 6. Cells transfected with EGFP-UBF but not deletion mutants show retarded incorporation of rhodamine-labelled dUTP in an in situ replication assay. Cells were infected with adenovirus and simultaneously transfected with various plasmids as indicated. After 18 hours the cells were assayed for incorporation of Rho-dUTP as outlined in the Materials and Methods. In each panel the fusion protein is in green and the labelled DNA is in red. (A) Two cells infected with adenovirus expressing different amounts of EGFP-UBF and labelled with Rho-dUTP. (B) The same cells, but with the sensitivity of the photodetectors raised to reveal the faint labelling. (C) A group of infected cells, some of which are expressing the deletion mutant UBF1-192EGFP, but with the microscope laser power and photo detector settings the same as in A for comparison. All images are of a single focal plane approximately 0.3 µm depth; bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 7. Antiserum to UBF retards viral DNA replication in permeabilised cells. (A) Viral DNA recovered from a number of replication assays, digested with KpnI and separated by electrophoresis and stained with ethidium bromide. The antiserum added to each assay is indicated above each lane, the size of the DIG-labelled molecular mass markers is indicated on the right hand side of the gel and each viral DNA fragment is labelled a-i on the left hand side of the gel. In addition, the DNA fragments corresponding to the left- and right-most ends of the viral DNA are marked with arrows. (B) The levels of DIG-labelled nucleotides incorporated into the DNA fragments. This was done by Southern transfer of the gel in A to nylon membrane and the DIG label detected as described in the Materials and Methods. (C) The same as in B but with a longer exposure. Below C is a schematic of the viral genome indicating where KpnI cuts the viral genome and the position of DNA fragments on the genome.

View larger version (64K): [in a new window]   Fig. 8. Adenovirus infection causes UBF to separate from RNA pol I and the major sites of RNA synthesis. (A) UBF (in green) and RNA synthesis (detected by FU labelling for 20 minutes as outlined in the methods and shown in red) in uninfected cells. (B) As A, but in adenovirus-infected cells at 18 hours post-infection. C shows the location of UBF (green) and RNA pol I (red) in uninfected cells, and D shows the same in adenovirus-infected cells at 18 hours post-infection. (E) Adenovirus-infected cells were FU labelled as before and simultaneously probed for RNA synthesis (green), RNA Pol I (red) and viral DBP (blue). (F) As for E, but probed for UBF (green), RNA pol I (red) and FU (blue). All images are of a single focal plane approximately 0.3 µm depth; bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 9. Adenovirus infection does not lead to a marked downturn in rRNA synthesis. Total RNA was extracted from infected HeLa cells at the times post-infection indicated, along with 2 mock-infected control samples taken at 8 hours post-mock infection (Con1) and 30 hours post-mock infection (Con2). (A) Each sample was assayed for rRNA synthesis by S1 nuclease protection assay for the 5' end of pre-rRNA. (B) Total RNA present in each sample, demonstrating similar quantities of RNA were present in each S1 nuclease protection assay.