TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin

Abstract

Human papillomavirus type 16 (HPV16) E2 regulates transcription from and replication of the viral genome, in association with viral and cellular factors. HPV16 E2 interacts functionally with TopBP1, a cellular protein essential for the initiation of cellular, and potentially viral, DNA replication. This report demonstrates that the absence of TopBP1 results in the redistribution of HPV16 E2 into an alternative cellular protein complex, resulting in enhanced affinity for chromatin. This redistribution does not significantly alter the ability of HPV16 E2 to either activate or repress transcription. We also show colocalization of both proteins on chromatin at late stages of mitosis, suggesting that TopBP1 could be the mitotic chromatin receptor for HPV16 E2. The possible significance of the results for the regulation of the viral life cycle is discussed.

FIG. 1.

TopBP1 depletion modifies E2 subcellular localization. (A) One microgram of the indicated reporter plasmid was cotransfected with increasing amounts of HPV16 E2 and 1 μg of depletion or mock-depletion plasmid. Cells were harvested 60 h posttransfection and assayed for luciferase activity (Promega). Luciferase activity for lysates containing no E2 was normalized to 1 and other activities were calculated as differences (n-fold). (B) Thirty-microgram portions of lysates from cells treated as described for panel A were Western blotted and probed for TopBP1 (upper gel) and HPV16 E2 (lower gel). (C) Outline of fractionation used for results shown in panel D. Cells were cotransfected with 1 μg HPV16 E2 and 1 μg depletion or mock-depletion plasmid and harvested 60 h later. The nuclei were prepared and pelleted out from the cytoplasm. The nuclei were then lysed and the nucleosol was separated from the insoluble pellet. Samples were Western blotted and probed for TopBP1, HPV16 E2, and ORC2 (predominantly nuclear marker). C, cytoplasmic; N, nuclear; P, pellet.

FIG. 2.

TopBP1 depletion causes association of E2 with less-soluble biochemical fractions. (A) Outline of salt extraction method. One microgram E2 (HPV16 E2 or BPV1 E2, as indicated for panels B and D) was cotransfected with 1 μg of either TopBP1 depletion or mock-depletion vector, and cells were harvested 60 h later. Cells were lysed in CSK buffer containing 0.5% Triton X-100, and the insoluble material was pelleted. This pellet was then serially extracted with increasing concentrations of NaCl in CSK (11), and all fractions were Western blotted. (Outline adapted from Kurg et al. [11].) (B) Shown are Western blots of HPV16 E2 fractionated as shown in panel A. The upper gel shows E2 from cells untreated with depletion vectors, the middle gel shows E2 from cells transfected with mock-depletion vector, and the lower gel shows E2 from cells transfected with depletion vector. (C) Shown are Western blots of HPV16 E2 fractionated as shown in panel A. Instead of undergoing TopBP1 depletion, cells were treated with hydroxyurea (HU) for 15 h prior to harvest (lower gel) or left untreated (upper gel). (D) Shown are Western blots of BPV1 E2 fractionated as shown in panel A. Cells were treated with mock-depletion plasmid (upper gel) or depletion plasmid (lower gel). S, soluble; P, pellet.

FIG. 3.

There are two pools of E2 in the cell. (A) Outline of fractionation method used for results shown in panel B. One microgram HPV16 E2 was cotransfected with 1 mg of either mock-depletion or depletion vector. Cells were harvested at 60 h posttransfection and cycloheximide was added to cells at 1-h intervals for the 6 h preceding harvest. The cells were then fractionated and salt extracted as described in the legend for Fig. 2A. (B) Shown are Western blots of HPV16 E2 fractions over a 6-h time course after cycloheximide addition. The left-hand panels show E2 from cells transfected with mock-depletion plasmid, and the right-hand panels show E2 from cells transfected with depletion plasmid. (C) Outline of fractionation method used for results shown in panel D. One microgram HPV16E2 was cotransfected with 1 μg of either depletion or mock-depletion plasmid, and cells were harvested 60 h later. The cells were lysed (50 mM Tris [pH 8], 400 mM NaCl, 0.5% NP-40), and a low-speed supernatant was prepared and loaded onto a sucrose gradient (5 to 20%). This was spun in an ultracentrifuge (120 × g; 15 h; 4°C) and then fractionated. The protein was precipitated using trichloroacetic acid and then Western blotted. (D) Shown are Western blots of HPV16 and TopBP1 from sucrose gradient fractions. The upper gel shows fractions from mock-depleted cells, and the lower gel shows fractions from depleted cells.

FIG. 4.

(A) TopBP1 depletion has the same effects when using a different knockdown system and in a different cell line. 293T and C33a cells were cotransfected with 1 μg HPV16 E2 and either siRNA plasmids (pSUPER, pSUPER-TopBP1) or siRNA oligonucleotides (anti-luciferase, anti-TopBP1) as indicated. The cells were harvested, and the lysates prepared and Western blotted. Blots were probed for TopBP1 to detect knockdown (upper gels), γ-tubulin as a loading control (middle gels), and HPV16 E2 to determine the effect of TopBP1 knockdown (lower gels). In both cell lines and with both siRNA systems, E2 is increased in the TopBP1-depleted lysates. (B) TopBP1 and E2 colocalise on the chromatin and centrosomes in the late telophase. U2OS cells stably transfected with E2 were fixed and stained with TVG261 mouse anti-HPV16 E2 (1:50) and with R1180 rabbit anti-TopBP1 (1:2,000) antibodies according to the protocol described in reference . The DNA was stained with 4′,6′-diamidino-2-phenylindole.