CIN612-9E cells were derived from a cervical lesion and contain hundreds of copies of extrachromosomally replicating HPV31 genomes [<b>52</b>]<b>.</b>

<p>These cells can be induced to differentiate with high calcium–containing medium, which switches on vegetative viral DNA replication <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003321#ppat.1003321-Moody1" target="_blank">[3]</a>. Many of these cells contain multiple small replication foci <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003321#ppat.1003321-Moody1" target="_blank">[3]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003321#ppat.1003321-Gillespie1" target="_blank">[4]</a>; but numerous cells contain one large foci, as shown here, perhaps indicative of a temporal evolution. The nucleus shown has been stained with DAPI (grey) and antibodies to γH2AX to identify the viral replication foci (shown in cyan), and RAD51 to identify centers of homologous recombination (shown in red). 3D reconstruction of Z-stacks of confocal images was performed using Bitplane Imaris.</p

The papillomavirus life cycle is closely coupled with differentiation of the host epithelium.

<p>The virus infects the dividing basal cells through a microabrasion. The viral DNA is maintained at a low copy number in these cells. When basal cells divide, some daughter cells move up in the epithelium and begin the process of terminal differentiation. Papillomaviruses are finely tuned to this process and turn on late transcription, translation, and late DNA replication in specific stages of the differentiation process. Vegetative viral DNA replication takes place in cells that are in either the G2 phase of the cell cycle or have exited the cell cycle. By inducing the DNA damage response and homologous recombination repair pathways, the virus can efficiently replicate progeny genomes in differentiated cells without competition from host DNA synthesis.</p

Salt extraction of cells expressing HPV16 E1 and E2 show that the E1 protein binds most tightly to host nuclei.

<p>Human keratinocytes were cotransfected with expression vectors for HPV16 E1 (pMEP9-E1), HPV16 E2 (pMEP4-E2), or both proteins. Cells were either fixed directly in paraformaldehyde (PFA), or proteins were extracted from cells in buffers containing either 100 mM or 300 mM NaCl prior to fixation in PFA. <b>A.</b> E1, E2 and endogenous Brd4 proteins were detected by indirect immunofluorescence. Staining for the E1 protein is shown in green, the E2 protein in cyan, and Brd4 in red. The dashed blue lines represent the perimeter of the nuclei (identified by DAPI staining, not shown). <b>B.</b> Level of E1, E2, and Brd4 were measured in individual cells extracted and fixed as described above using the Contour feature of Imaris software (Bitplane) to extract values from each channel in individual nuclei. Average values obtained from at least five reconstructed nuclei are shown and the error bars represent the standard deviation. Individual transfected cells could not be identified in cells transfected with the empty pMEP4/9 vectors or in cells transfected with HPV16 E2 vectors and extracted in 300 mM NaCl containing buffer. In these samples, random cells were selected for quantitation.</p

Properties of replication foci.

1<p>differentiated 9E cells contain a very heterogeneous mix of cell and foci phenotypes and it is difficult to determine more subtle effects on the distribution of different foci types using this method.</p

In the presence of a replicating viral origin, the size of E1/E2 foci increases and Brd4 is displaced.

<p><b>A.</b> Human keratinocytes were cotransfected with expression vectors for HPV16 E1 and E2 in the presence of a plasmid containing the minimal HPV16 replication origin, p16ori (+ ori) or a control plasmid without the origin, pKS (− ori). The E1 and Brd4 proteins were detected by immunofluorescence. Representative foci from each condition are shown. The diameter of E1 foci was measured using Bitplane Imaris. Error bars represent the standard error of the mean. <b>B.</b> Human keratinocytes were cotransfected with expression vectors for HPV16 E1 and E2 in the presence of a plasmid containing the minimal HPV16 replication origin, p16ori (+ ori) or a control plasmid without the origin, pKS (− ori). E1 and E2 expression was induced four hours before fixation in the presence of 25 µg/ml AraC and E1, E2 and Brd4 proteins were detected by immunofluorescence. Representative cells from each condition are shown. The diameter of E1 foci was measured using Bitplane Imaris. Error bars represent the standard error of the mean.</p

Brd4 surrounds replication foci in differentiating cells harboring HPV31 genomes.

<p><b>A and B</b> Replication foci were detected by FISH for HPV31 DNA in uninfected keratinocytes (i), and undifferentiated (ii) and differentiated (iii and iv) CIN-612 9E cells. <b>C and D</b> Replication foci were detected by immunofluorescence for γH2AX (cyan) and were also stained for Brd4 (red) DNA in uninfected keratinocytes (i), and undifferentiated (ii) and differentiated (iii and iv) CIN-612 9E cells. <b>A, B, C, D</b>. 3D image stacks were deconvolved and digitally reconstructed using Huygens Essential software. Volume images are shown in A and C and high resolution surface renderings of all objects were generated by Bitplane Imaris software and are shown in B and D. <b>E</b> The γH2AX and Brd4 proteins were detected by immunofluorescence in differentiated CIN-612 9E cells., and following a brief fixation, viral DNA was detected by FISH. Volume images are shown in i and high resolution surface renderings of all objects were generated by Bitplane Imaris software and are shown in ii, iii and iv. γH2AX is shown in cyan, Brd4 in red, and HPV31 DNA in green.</p

Homologous recombination marker, Rad51, colocalizes with HPV replication foci.

<p><b>A.</b> (i) Nuclear foci were generated by differentiation of CIN612 9E cells for five days. Cells were stained with antibodies against Brd4 (rabbit mcb2), Rad51 (mouse) and γH2AX (mouse). Isotype specific secondary antibodies were used to distinguish between the mouse antibodies. Cellular DNA is counterstained with DAPI (blue in merged panels). 3D image stacks were deconvolved using Huygens Essential software.  (ii) High resolution surface renderings of all objects generated by Bitplane Imaris software. (iii) High resolution surface rendering of the replication foci shown in (ii). (iv) Cross-section through the replication foci shown in (i, ii, iii). Rad51 and γH2AX are localized within the foci and are surrounded by small speckles of Brd4. <b>B.</b> Nuclear foci were generated by transfection of HPV16 E1 and E2 expression vectors (with and without p16ori). Cells were stained with antibodies against E1, Brd4 (rabbit mcb2) and Rad51 (mouse). The dashed blue lines represent the perimeter of the nuclei (identified by DAPI staining, not shown). <b>C.</b> The percentage of HPV16 E1/E2 foci containing Rad51, obtained from experiments as described in B, is represented by the blue bars. The percentage of γH2AX foci containing Rad51 in differentiated CIN612 9E cells (as described in A) is represented by the red bar. Error bars represent standard deviation.</p

Brd4 localizes to nuclear foci formed by HPV16 E1 and E2 proteins.

<p>Human keratinocytes were transfected with pMEP4/9 (empty), pMEP9-HPV16 E1 and pMEP4-HPV16 E2 expression vectors, as indicated. E1, E2 and endogenous Brd4 proteins were detected by indirect immunofluorescence. Staining for E1 proteins is shown in green, E2 proteins in cyan, and Brd4 in red. High resolution 3D images reconstructed by deconvolution of stacks of confocal images are shown. At least five cells were digitally reconstructed from each condition and a representative image is shown. The dashed blue lines represent the perimeter of the nuclei (identified by DAPI staining, not shown). <b>A.</b> Brd4 staining in a single optical slice through the center of the nucleus. <b>B.</b> An optical section through nuclei expressing E1, E2 or E1/E2. The crosshairs show the cut point through the x-, y-, and z-axes of the data set. These are the same cells shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003777#ppat-1003777-g001" target="_blank">Figure 1A</a>. <b>C.</b> An enlargement of a portion of the nuclei from the images shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003777#ppat-1003777-g001" target="_blank">Figure 1B</a>. The scale bar represents 1 µm.</p

Analysis of the E1–E2 functions required for localization of Brd4 to nuclear E1–E2 foci in the presence and absence of viral origin containing DNA.

<p><b>A.</b> Representation of mutations that specifically inactivate E1 and E2 functions. E1: K230A, S231A (defective in specific DNA binding); K483A (ATPase deficient). E2: E39A (deficient in interaction with E1); R37A, I73A (deficient in transcriptional regulation and Brd4 binding); R302K, R304K (DNA binding defective). <b>B.</b> Wild-type or mutated HPV16 E1 and E2 expression plasmids were transiently transfected into keratinoctytes and cells were stained by immunofluorescence for E1 (green), E2 (cyan) and Brd4 (red). The dashed blue lines represent the perimeter of the nuclei (identified by DAPI staining, not shown). The cells in the left panel were also transfected with a control plasmid (pKS) and those on the right with a minimal origin containing plasmid (p16ori). Fluorescent signals were collected at unsaturated levels, according to viral protein expression, and were later increased to enhance figure brightness. <b>C.</b> Cells expressing E1 and E2 in the presence or absence of origin containing DNA were counted and scored for the presence of foci that co-localized with Brd4.</p

Comparison of the characteristics of foci formed by expression of E1 and E2 proteins and naturally occurring replication foci in differentiated HPV containing cells.

<p>HFKs were transiently transfected with HPV16 E1 and E2 expression plasmids and the control plasmid pKS (left column), or with HPV16 E1 and E2 expression plasmids and the replication origin plasmid p16ori (middle column). HPV31 genome containing 9E cells (right column) were differentiated for five days as described in Methods. Cells were stained for E1, E2, Brd4, H3K4me1, H4K8ac and H3K56ac, Rad51, pATM and γH2AX, as indicated. The rabbit Brd4 mcb2 antiserum is used in most panels, except for H3K4me1, H4K8ac and H3K56ac staining in HPV31 9E cells (right column), where a mouse Brd4 antibody 8H2 was used. A goat anti-Rad51 antibody was also used in these panels. A mouse Rad51 antibody was used to stain HPV31 9E cells, along with Brd4 (rabbit mcb2) and γH2AX (mouse). Isotype specific secondary antibodies were used to distinguish between the mouse antibodies. Cellular DNA is counterstained with DAPI (blue in merged panels).</p