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. 2005 Feb;79(4):2474-83.
doi: 10.1128/JVI.79.4.2474-2483.2005.

Adenovirus protein VII functions throughout early phase and interacts with cellular proteins SET and pp32

Yuming Xue  1 , Jeffrey S Johnson, David A Ornelles, Judy Lieberman, Daniel A Engel
Affiliations

Adenovirus protein VII functions throughout early phase and interacts with cellular proteins SET and pp32

Yuming Xue et al. J Virol. 2005 Feb.

Abstract

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. "Input" protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.

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Figures

FIG. 1.
FIG. 1.
Appearance of input protein VII and new synthesis of pre-VII and mature protein VII during infection. HeLa cells were infected for the indicated times and analyzed by Western blot for protein VII (A and B) or the 72-kDa DBP (C). The anti-protein VII antibody recognizes both pre-VII (pVII) and protein VII. M, mock infection; C, control lane containing virus-derived protein VII.
FIG. 2.
FIG. 2.
Protein VII association with viral chromatin. (A) HeLa cells were infected for the indicated times and analyzed by ChIP as described in Materials and Methods. Cells were treated with formaldehyde to cross-link protein to nucleic acid. Cell extracts were prepared, sonicated to shear the DNA to roughly 200 bp, and then incubated with anti-protein VII antibody (VII) or control preimmune serum (C). Precipitates were heated to reverse the cross-links, deproteinized, and assayed by PCR with primers specific for the E1B gene. G, genomic virus DNA control. pi, postinfection. (B) ChIP efficiency. Cells were infected for 4 h and analyzed by ChIP.
FIG. 3.
FIG. 3.
α-Amanitin treatment of infected cells. (A) Cells were infected with dl309 for 4 h and treated with 10 μg of α-amanitin/ml starting 1 h postinfection. (A) ChIP analysis was performed as described in Materials and Methods by using primers representing the L3 and E1B coding regions. C, control preimmune serum; VII, anti-protein VII antiserum. (B) Western blot analysis of E1A and input protein VII. Ramps indicate that increasing amounts of extract were analyzed.
FIG. 4.
FIG. 4.
(A) ChIP of infected cells treated with HU. Cells were infected with dl309 for the indicated times and treated with 3 μg of HU/ml starting 1 h post infection (pi). ChIP was performed as described in Materials and Methods by using primers specific for the L3 gene. C, control preimmune serum; VII, anti-protein VII antibody. (B) Cells were infected for the indicated times and treated with HU as described for panel A. Extracts were prepared and analyzed by SDS-PAGE and Western blotting for DBP.
FIG. 5.
FIG. 5.
Viral chromatin and protein VII association with pp32 and SET. (A) ChIP analysis. Cells were infected with dl309 for 6 h and analyzed by ChIP with anti-pp32 (α-pp32), anti-SET (α-SET), or control (C) antibodies. PCR products: MLP, major late promoter; L3, late region 3; E1A, early region 1A. (B) HeLa cell lysates were used in binding reactions with GST-protein VII, GST-E1A289, or the unrelated protein control GST-CAS YXXP (7). Bound proteins were separated by SDS-PAGE and analyzed by Western blotting for SET or pp32. Lower panel, structure of GST-protein VII mutants. Bars indicate regions of protein VII expressed in the mutants. The GST moiety is not illustrated.
FIG. 6.
FIG. 6.
Protein VII is in nuclear dots in early phase. HeLa cells were grown on glass coverslips and infected with phenotypically wild-type dl309 for 4 h. Cells were fixed by cold methanol dehydration and probed with rabbit polyclonal anti-protein VII antibody. For visualization, cells were incubated with anti-rabbit rhodamine secondary antibody conjugate. Prior to mounting, cells were counterstained with DAPI. Phase, phase-contrast microscopy; mock, uninfected control; inf, infected cells.
FIG. 7.
FIG. 7.
Protein VII dot localization throughout infection. HeLa cells were infected for the indicated times at a MOI of 20. Cells were fixed by cold methanol dehydration and probed with mouse monoclonal anti-DBP and rabbit polyclonal anti-protein VII antibodies. For visualization, cells were probed with anti-mouse FITC and anti-rabbit rhodamine secondary antibody conjugates. Prior to mounting, cells were counterstained with DAPI.
FIG. 8.
FIG. 8.
Protein VII dot localization at late times. HeLa cells were infected for the indicated times at a MOI of 20. Cells were fixed by cold methanol dehydration and probed with mouse monoclonal anti-DBP and rabbit polyclonal anti-protein VII antibodies. For visualization, cells were probed with anti-mouse FITC and anti-rabbit rhodamine secondary antibody conjugates. Prior to mounting, cells were counterstained with DAPI. Arrowheads indicate examples of cells containing replication centers.
FIG. 9.
FIG. 9.
Disappearance of nuclear dots requires RNA synthesis but not DNA synthesis. HeLa cells were infected for the indicated times and were treated at 1 h postinfection with either 25 μg of Ara-C/ml or 10 μg of α-amanitin/ml. Cells were fixed by cold methanol dehydration and probed with mouse monoclonal anti-DBP and rabbit polyclonal anti-protein VII antibodies. For visualization, cells were probed with anti-mouse FITC and anti-rabbit rhodamine secondary antibody conjugates. Prior to mounting, cells were counterstained with DAPI.
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