The human papillomavirus type 8 E2 tethering protein targets the ribosomal DNA loci of host mitotic chromosomes

Abstract

For many papillomaviruses, the viral protein E2 tethers the viral genome to the host mitotic chromosomes to ensure persistent, long-term maintenance of the genome during cell division. Our previous studies of E2 proteins from different genera of papillomaviruses have shown that they bind to different regions of the host chromosomes during mitosis. For example, bovine papillomavirus type 1 (BPV-1) E2 binds to all chromosomes as small speckles in complex with the cellular protein Brd4. In contrast, the human papillomavirus type 8 (HPV-8) E2 protein binds as large speckles at the pericentromeric regions of chromosomes. Here we show that these speckles do not contain Brd4, and unlike that of BPV-1, the N-terminal Brd4-interacting domain of HPV-8 E2 is not required for chromosome binding. In contrast to BPV-1 E2, the HPV-8 E2 protein targets the short arms of acrocentric mitotic chromosomes. Furthermore, the E2 protein interacts with the repeated ribosomal DNA genes found in this location and colocalizes with UBF, the RNA polymerase I transcription factor. Therefore, HPV-8 E2 genome tethering occurs by a Brd4-independent mechanism through a novel interaction with specific regions of mitotic chromosomes. Thus, a wide range of viruses have adopted the strategy of linking their genomes to host chromosomes, but individual viruses use different chromosomal targets. Characterization of these targets will enable the development of antiviral therapies to eliminate the viral genomes from infected cells.

FIG. 1.

BPV-1 and HPV-8 E2 proteins bind to different chromosomal locations. BPV-1 E2 (green) and HPV-8 E2 (red) proteins were detected in CV-1 cells expressing both proteins by indirect immunofluorescence, using B201 and anti-FLAG antibodies, respectively. The kinetochore was detected with an anticentromere antibody (aqua blue). Cellular DNA was counterstained with DAPI (blue).

FIG. 2.

Both full-length and HC HPV-8 E2 proteins bind mitotic chromosomes. (A) Diagram showing the structure of wild-type (WT) HPV-8 E2 and the individual domains used for chromosome localization. N, H, and C represent the N-terminal, hinge, and C-terminal domains, respectively. The amino acid positions delineating these domains are shown. (B) Immunolocalization of HPV-8 E2 proteins and derived domains expressed in CV-1 cells. HPV-8 E2 proteins were detected with anti-FLAG antibody (green), and cellular DNA was counterstained with DAPI (blue). neg, negative. (C) Alignment of the amino acid sequences of the BPV-1 and HPV-8 E2 proteins.

FIG. 3.

Interaction of HPV-8 E2 protein in the absence of Brd4 binding. (A) In vitro-translated E2 proteins were tested for their abilities to bind Brd4. Aliquots (10 μl) of E2-containing reticulocyte lysate (adjusted for the concentration of E2) were assayed for binding to 5 μl of Brd4 protein extract prebound to anti-FLAG immunobeads. Bound E2 proteins (B) were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. The input lanes (I) contain a 1/5 volume of lysate added to the binding reactions. Molecular size markers are at the right. (B) Percentage of input protein bound for each E2 protein in the presence or absence of Brd4 protein. wt, wild type. (C) Immunolocalization of HPV-8 E2 proteins and Brd4 on mitotic chromosomes in CV-1 cells. E2 was detected with anti-FLAG antibody (green) and Brd4 with anti-Brd4 antiserum (red). DNA was counterstained with DAPI (blue). WT, wild type; neg, negative.

FIG. 4.

Distribution of HPV-8 E2 protein on mitotic chromosomes. (A) Immunolocalization of E2 protein and centromeric ACA antigen on a metaphase chromosome spread of C33A cells expressing HPV-8 E2. E2 was detected with an anti-FLAG antibody (green), and centromeres were detected with an anti-ACA centromeric antibody (red). DNA was counterstained with DAPI (blue). (B) Images of isolated, individual chromosomes showing HPV-8 E2 and ACA staining.

FIG. 5.

HPV-8 E2 associates with the short arms of acrocentric chromosomes. (A, B, and C) HPV 8 E2 was mapped to acrocentric chromosomes by combined immunostaining of E2 (green) and FISH, using α-satellite probes (red). Cellular DNA is counterstained with DAPI and shown in blue. (D) FISH, with α-satellite probes specific for nonacrocentric chromosomes 1, 5, and 19, demonstrates the specificity of the association of HPV-8 E2 with acrocentric chromosomes.

FIG. 6.

β-satellite DNA and HPV-8 E2 occupy adjacent, but distinct, regions on the short arms of acrocentric chromosomes. (A) Images of individual chromosomes showing localization of E2 and β-satellite DNA. Combined immunofluorescence with anti-FLAG antibody was used to detect E2 protein (green), and FISH was used to detect β-satellite DNA (red) on metaphase spreads. DNA is counterstained with DAPI (blue). (B) An E2 binding site is located between the centromere and β-satellite DNA. Metaphase chromosome spreads with E2 (green), centromere (cyan blue), and β-satellite DNA (red) are shown. (C) Image of an individual chromosome with E2 (green), β-satellite DNA (red), and centromere (cyan blue) fluorescence signals.

FIG. 7.

HPV-8 E2 colocalizes with rDNA on mitotic chromosomes. (A) Combined immunofluorescence and FISH were used to detect E2 (green), rDNA (red), and ACA centromere antigen (cyan blue) on metaphase chromosome spreads of HPV-8 E2-expressing C33A cells. DNA is shown in blue. (B) E2, rDNA, and ACA staining on randomly chosen isolated chromosomes from different metaphase chromosome spreads. (C) A fluorescence intensity light scan was obtained by drawing a line through the three fluorescence signals of E2, rDNA, and ACA. The chromosome used for drawing the line scan is shown in the inset.

FIG. 8.

HPV-8 E2 and UBF1 colocalize on mitotic chromosomes. (A) E2 (green) and UBF1 (red) were detected in metaphase chromosome spreads by indirect immunofluorescence, using anti-FLAG and anti-UBF antibodies, respectively. (B) Localization of E2 and UBF relative to the centromere was determined by immunostaining with ACA (aqua blue), E2 (green), and UBF (red) antibodies. DNA was stained with DAPI (blue). (C) Fluorescence intensity light scan of E2, UBF1, and ACA signals. The chromosome used for drawing the line scan is shown in the inset.

FIG. 9.

Schematic representation of E2 localization. Localization of E2 (green), rDNA (red), UBF (red), β-satellite DNA (red), and centromere DNA (aqua blue) on acrocentric chromosome. Colocalization of the E2 signal (green) and rDNA/UBF (red) is represented as yellow.