BRD4S interacts with viral E2 protein to limit human papillomavirus late transcription

Abstract

The E2 protein encoded by human papillomaviruses (HPV) is a sequence-specific DNA-binding protein that recruits viral and cellular proteins. Bromodomain-containing protein 4 (BRD4) is a highly conserved interactor for E2 proteins that has been linked to E2's functions as transcription modulator, activator of viral replication and segregation factor for viral genomes. In addition to BRD4, a short form of BRD4 (BRD4S) is expressed from the BRD4 gene which lacks the C-terminal domain of BRD4. E2 proteins interact with the C-terminal motif (CTM) of BRD4, but a recent study suggested that the phospho-dependent interaction domain (PDID) and the basic interaction domain (BID) in BRD4 also bind to E2. These domains are also present in BRD4S. We now find that HPV31 E2 interacts with the isolated PDID domain in living cells and also with BRD4S which is present in detectable amounts in HPV-positive cell lines and is recruited into HPV31 E1 and E2 induced replication foci. Overexpression and knockdown experiments surprisingly indicate that BRD4S inhibits activities of E2. In line with that, the specific knockdown of BRD4S in the HPV31-positive CIN612-9E cell line induces mainly late viral transcripts. This occurs only in undifferentiated but not differentiated cells in which the productive viral replication cycle is induced. These data suggest that the BRD4S-E2 interaction is important to prevent HPV late gene expression in undifferentiated keratinocytes which may contribute to immune evasion and HPV persistence.ImportanceHuman papillomaviruses (HPV) have coevolved with their host by using cellular factors like bromodomain-containing protein 4 (BRD4) to control viral processes such as genome maintenance, gene expression and replication. We here show that, in addition to the C-terminal motif in BRD4, the phospho-dependent interaction domain in BRD4 interacts with E2 proteins which enable the recruitment of BRD4S, the short isoform of BRD4, to E2. Knock-down and overexpression of BRD4S reveals that BRD4S is a negative regulator of E2 activities. Importantly, the knockdown of BRD4S induces mainly L1 transcripts in undifferentiated CIN612-9E cells, which maintain replicating HPV31 genomes. Our study reveals an inhibitory role of BRD4S on HPV transcription, which may serve as an immune escape mechanism by the suppression of L1 transcripts and thus contribute to the establishment of persistent HPV infections.

FIG 1

Interaction of BRD4 and HPV31 E2 in a flow cytometry-based FRET assay. (A) Schematic depiction of BRD4 (aa 1 to 1362) and BRD4S (aa 1 to 722) and their functional domains as follows: BD1 (bromodomain 1), PDID (phosphorylation-dependent interaction domain), BD2 (bromodomain 2), NPS (N-terminal cluster of phosphorylation sites), BID (basic residue-enriched interaction domain), ET (extra terminal domain), and CTM (C-terminal motif). BRD4S contains the unique residues GPA at positions 720 to 722 compared to BRD4. Additionally, the schematic depiction of HPV31 E2 protein (aa 1 to 372) showing the transactivation domain (TAD), hinge region, and DNA-binding/dimerization domain (DBD). (B) Gating strategy for fluorescence-activated cell sorter (FACS)-FRET analysis and BFP-31 E2 domain expression and nuclear localization. (A) One experiment is shown representatively for C33A cells, which were not transfected (mock) or transfected with expression vectors for SYFP, BFP, SYFP-BFP fusion, or BRD4-SYFP and BFP-HPV31 E2. Living cells were analyzed 48 h after transfection for FRET-positive signals using MACSQuant VYB (Miltenyi Biotec). Living cells were gated for double positive cells by excitation with the 405-nm laser to measure BFP emission with the 450/50-nm band-pass filter and by excitation with the 488-nm laser to measure SYFP emission by 525/50-nm band-pass filter (lane 1). The next gate ensures that SYFP-expressing cells, which emit a YFP signal (488/525) and a FRET signal measured by 525/50-nm filter that resulted in excitation with the 405-nm laser, are excluded to avoid false-positive cell count (lane 2). The last gate includes cells that emit a BFP signal (405/450) and a FRET signal, which was generated by the energy transfer of the BFP (excited by 405-nm laser) to SYFP measured by 525/50-nm filter. (C) C33A cells were transfected with expression vectors for SYFP, BFP, SYFP-BFP fusion, or combinations of BRD4-SYFP or BRD4dCTM-SYFP and BFP-HPV31 E2 or BFP-HPV31 E2 I73L. Living cells were analyzed 48 h after transfection for FRET-positive signals using MACSQuant VYB (Miltenyi Biotec). Mean values of FRET-positive cells (%) from at least three independent experiments are shown. Error bars indicate the standard error of the mean (SEM). Statistical significance was determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple-comparison test (compared to BRD4-SYFP + BFP-31 E2). **, P < 0.01; ***, P < 0.001. (D) Immunoblot of transfected C33A cells using antibodies specific for SYFP to detect BRD4 fusions or BFP to detect HPV31 E2 fusion proteins. HSP90 was used as a loading control. (E) C33A cells were transfected with combinations of BFP-HPV16 E2 or HPV31 E2 and SYFP-BRD4-PDID, SYFP-BRD4-BID, or SYFP-BRD4-CTM expression vectors, and FRET-positive cells were determined as described above. Mean values of FRET-positive cells (%) from at least three independent experiments are shown. Error bars indicate the SEM. (F) C33A cells were transfected with 500 ng of BFP-31 E2 TAD-hinge, BFP-31 E2 hinge, BFP-31 E2 hinge-DBD vectors. Whole-cell lysates were analyzed by immunoblot using an antibody specific for BFP. HSP90 was used as loading control. C33A cells transfected with 50 ng of expression vectors for BFP-31 E2 TAD-hinge, BFP-31 E2 hinge, and BFP-31 E2 hinge-DBD were analyzed by immunofluorescence with an antibody specific for BFP. DAPI was used to visualize DNA. (G) C33A cells were transfected with a total of 1 μg of expression vectors for SYFP-BRD4-PDID, -BID, -CTM, and BFP-HPV31 E2 TAD-hinge, BFP-HPV31 E2 hinge, and BFP-HPV31 E2 hinge-DBD. Mean values of FRET-positive cells (%) from three independent experiments are shown, and error bars indicate SEM.

FIG 2

Several domains in PDID are required for interaction with E2. (A) C33A cells were cotransfected with SYFP-BRD4 PDID, -PDIDd287-348, -PDIDdBD2, -PDIDd462-482, -PDIDdNPS, or -PDIDd505-530 and BFP-HPV31 E2 expression vectors. Mean values of FRET-positive cells (%) are derived from three independent experiments, and error bars indicate the SEM. (B) BFP-BRD4-PDID, -BID, -CTM were tested in different combinations with SYFP-BRD4-PDID, -BID, -CTM in C33A cells. Additionally, the interactions of BFP-BRD4-PDID with SYFP-BRD4-PDID, -PDIDdBD2, -PDIDdNPS, and -PDIDd505 were analyzed. Mean values of FRET-positive cells (%) are derived from three independent experiments, and error bars indicate the SEM. Statistical significance was determined by one-way ANOVA and Dunnett’s multiple-comparison test (compared to SYFP-BRD4-PDID + BFP-HPV31 E2 [A] or SYFP-BRD4-PDID + BFP-BRD4-PDID [B]); ns, P > 0.05; **, P < 0.01; ****, P  < 0.0001. (C) Immunoblot analyses of C33A cells transfected with expression vectors for SYFP-BRD4-PDID, -BID, -CTM, -PDIDdBD2, -PDIDdNPS, or -PDID d505-530. Untransfected cells served as a negative control (C33A). HSP90 was used as loading control.

FIG 3

BRD4S interacts with HPV31 E2 and is expressed in different keratinocyte cell lines. (A) C33A cells were transfected with 10 μg of expression vectors for HA-tagged HPV31 E2 and Flag-tagged BRD4, BRD4S, or BRD4dCTM proteins. Whole-cell lysates were directly analyzed (input) or precipitated with magnetic anti-Flag beads. Lysates were analyzed using α-HA and α-Flag antibodies. (B) Immunoblot analysis of endogenous BRD4S expression in whole-cell extracts of normal human keratinocytes (NHK), C33A, and CIN612-9E cells. To detect BRD4S, α-BRD4 (N-terminal) and for BRD4 detection, α-BRD4 (C-terminal) antibodies were used. Alpha-tubulin was used as a loading control.

FIG 4

BRD4S colocalizes with E1/E2 replication foci. (A) C33A cells were transfected with expression vectors for HPV31 E1 (500 ng; pCMV neo 31 E1 – 3×Flag), HPV31 E2 (50 ng; pSX31 E2–HA), BRD4 (700 ng; pcDNA3-flag BRD4), or BRD4S (500 ng; pcDNA3-flag BRD4S) and analyzed by immunofluorescence with anti-HA or anti-Flag antibodies. DAPI was used to stain DNA. (B) C33A cells were transfected with expression vectors for HPV31 E1 (flag) and E2 (HA) and with or without 500 ng pGL31URR-Luc (31 URR) and stained with anti-HA, anti-Flag, and DAPI. (C, D) C33A cells were transfected with combinations of expression vectors for HPV31 E1 (untagged), HPV31 E2 (HA), BRD4 (flag), BRD4S (flag), or URR 31 (pGL31 URR) and stained with anti-HA, anti-Flag, and DAPI. (E) Quantification of the levels of colocalization between E2 and BRD4 or BRD4S. Data are expressed as the Pearson correlation coefficient between E2 and BRD4(S) signals. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple-comparison test (ns, not significant; ***, P < 0.001; ****, P < 0.0001).

FIG 5

BRD4S inhibits E2 transactivation. (A) C33A cells were transfected with 0.5 ng pCMV-Gluc, 50 ng pC18-Sp1-Luc (structure shown above the graph), 10 ng of BFP-31 E2, and 25, 50, or 100 ng of SYFP-BRD4S or empty SYFP and BFP vectors. Values were calculated as the ratio of firefly to Gaussia luciferase and are shown relative to the activity of the reporter in the presence of BFP and SYFP expression vectors. Mean values are derived from at least three independent experiments, and error bars indicate the SEM. Statistical significance was determined by one-way ANOVA and Dunnett’s multiple-comparison test (compared to activity of reporter with HPV31 E2); ns, P > 0.05; *, P < 0.05. (B) Immunoblot of untransfected C33A cells or which were transfected with 100 ng of BFP-31 E2 and 250, 500, or 1,000 ng of SYFP-BRD4S or/and empty SYFP and BFP vectors using antibodies specific for SYFP to detect SYFP and BRD4S expression or BFP to detect BFP and HPV31 E2. HSP90 was used as a loading control. (C) C33A cells were transfected with 0.5 ng pCMV-Gluc, 50 ng pGL31 URR-luc (structure shown above the graph), 100 ng HPV31 E1, 10 ng of BFP-31 E2, and 25, 50, or 100 ng of SYFP-BRD4S or empty pSG5, SYFP, or BFP vectors. Values were calculated as the ratio of firefly to Gaussia luciferase and are shown relative to the activity of the reporter in the presence of pSG5-, BFP-, and SYFP expression vectors. Mean values are derived from at least three independent experiments, and error bars indicate the SEM. Statistical significance was determined by one-way ANOVA and Dunnett’s multiple-comparison test (compared to activity of reporter with HPV31 E2 and HPV31 E1); *, P < 0.05; **, P < 0.01.

FIG 6

siRNA-mediated knockdown of BRD4S enhances E2-dependent reporter activity. (A) C33A cells were transfected with siRNA (90 pmol) against BRD4S, and 72 h after transfection, total RNA was analyzed for BRD4 and BRD4S expression using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene. Results are presented relative to siRNA control (=1) and represent the mean of three independent experiments. Error bars indicate the SEM. (B) Expression of BRD4S in C33A cells transfected with control siRNA (siCon) or siBRD4S was analyzed by immunoblot 72 h after transfection using α-BRD4 (N-ter) for BRD4S detection and α-BRD4 (C-ter) for BRD4 detection. HSP90 was used as reference. The graphs represent the quantification of signals for BRD4, BRD4S, and HSP90 from three independent experiments, and the error bars indicate the SEM. Statistical significance was determined by paired t test (ns, not significant; *, P < 0.05). (C) C33A cells were transfected with control siRNA (Con) or siBRD4S (15 pmol). One day later, cells were transfected with 0.5 ng pCMV-Gluc, 50 ng of pC18-Sp1-Luc, 10 ng of BFP-31 E2, and either 100 ng of Flag-tagged BRD4S or 100 ng of empty vector (pcDNA3.1). Mean values represent ratios of firefly luciferase to Gaussia luciferase activity relative to the E2-induced reporter activity from at least three independent experiments, and error bars indicate the SEM. Statistical significance was determined by one-way ANOVA and Dunnett’s multiple-comparison test; ****, P < 0.0001.

FIG 7

siRNA knockdown of BRD4S increases HPV31 late gene expression. (A) CIN612-9E cells were transfected with 90 pmol of control siRNA or siBRD4S, and 72 h after transfection, whole-cell lysates from undifferentiated or differentiated cells were analyzed by immunoblot analysis using α-BRD4 (N-ter) for BRD4S detection and α-BRD4 (C-ter) for BRD4 detection. HSP90 was used as reference. (B) Total RNA of CIN612-9E cells transfected with 90 pmol of control siRNA or siBRD4S was analyzed for BRD4, BRD4S, and the HPV31 transcripts E4^L1, E1^E4, E8^E2, and E6*. As reference, gene GAPDH was used. Mean values are derived from three independent experiments and are presented relative to siRNA control (=1). (C) CIN612-9E cells transfected with 90 pmol of control siRNA or siBRD4S were differentiated the next day in 1.5% methylcellulose solution for 48 h. Total RNA was analyzed for BRD4, BRD4S, and the HPV31 transcripts E4^L1, E1^E4, E8^E2, and E6*. As reference, gene GAPDH was used. Mean values are derived from three independent experiments and are presented relative to undifferentiated siRNA control (siCon). Error bars indicate the SEM. Statistical significance was determined with a two-way ANOVA and Sidak’s multiple-comparison test; ****, P < 0.0001.

FIG 8

CRISPR/Cas9-mediated knockdown of BRD4S increases expression of the HPV31 E4^L1 transcript in undifferentiated cells. (A) Schematic depiction of BRD4S genomic structure (intron and exon 12) with gRNA sequences gBRD4SA and gBRD4SB, which disrupt the splice acceptor site (black). Additionally, the target site of siBRD4S is shown. (B) Immunoblot analysis of CIN612-9E whole-cell extracts, stably infected with pLentiCrispr v2 (empty vector), pLentiCrispr BRD4S-ex12-SA_A (A), or pLentiCrispr BRD4S-ex12-SA_B (B). p1 shows BRD4S expression in cells harvested directly after puromycin selection, and p2 shows the same cells after they were passaged once. To detect BRD4 and BRD4S, an α-BRD4 (N-terminal) antibody was used. HSP90 was used as reference. (C) Relative BRD4S expression from CIN612-9E cells infected with lentiviral pLentiCrispr v2 vector system analyzed by qPCR. p1 summarizes four independent experiments. p2 represents the mean of two independent experiments. GAPDH was used as a reference gene. Results are presented relative to the empty pLentiCrispr v2 vector (=1). (D) Total RNA of the four independent experiments from (p1) were analyzed for the expression of BRD4, BRD4S, and the HPV31 transcripts E4^L1, E1^E4, E8^E2, and E6* using GAPDH as a reference gene. Results are presented relative to the empty pLentiCrispr v2 vector (=1). Error bars indicate the SEM. Statistical significance was determined with a two-way ANOVA and Dunnett’s multiple-comparison test; *, P < 0.05.