Fig 1.
Proteomic analysis identify NCoR/SMRT complex components as E8-domain-dependent interactors of HPV16 and 31 E8^E2C proteins.
(A) Common interactors of HPV16 and HPV31 E8^E2C proteins identified by NanoLC-MS/MS analysis from anti-HA immunoprecipitates from 293T/HPV16 E8^E2C-HA, 293T/HPV31 E8^E2C-HA or 293T/pIRES-puro cells (PEP = posterior error probability). (B) Co-immunoprecipitation analysis reveals an interaction of wt HPV16 and 31 E8^E2C proteins with HDAC3, NCoR, SMRT, TBL1 and TBLR1 but not with E8^E2C KWK mt proteins. Cell lysates from stable 293T cell lines were directly analyzed (input) or precipitated with α-HA antibody (IP) and subjected to immunoblot analysis with the indicated antibodies.
Fig 2.
HPV1 and 8 E8- genomes replicate in normal human keratinocytes.
HPV1 and 8 wt or E8- genomes were transfected into normal human keratinocytes and low-molecular weight DNA was isolated 7d p.t. and analyzed by DpnI digestion and Southern blot. Linearized HPV1 or HPV8 genomes (100pg) were used as a molecular size marker (M). The positions of DpnI-resistant, replicated (rep.) and DpnI-sensitive, non-replicated (non-rep.) genomes are indicated.
Fig 3.
(A, B) HPV1 and E8^E2C repress transcription in an E8-domain dependent manner.
NHK cells were transfected with 300ng of the pC18-Sp1-luc firefly luciferase (Fluc) reporter and 10ng of the expression vectors for HPV1 E8^E2C or E8^E2C K2A d3-10 mt or with 1ng of the expression vectors for HPV8 E8^E2C or E8^E2C K2A d3-10 mt. HeLa cells and RTS3b cells were transfected with 100ng of the reporter plasmid and with the same amounts of the expression vectors as the NHK cells. Expression vectors for HA-tagged versions of HPV1 and 8 wt or mutant E8^E2C proteins were transfected into HeLa cells and nuclear extracts were subjected to immunoblotting and analyzed with α-HA and α-KRIP1 as a reference. (C) Comparative analysis of E8^E2C repressor activities. HeLa or RTS3b cells were transfected with 100ng pC18-Sp1-luc reporter plasmid and increasing amounts of E8^E2C expression vectors (0.1, 1 or 10ng). Differences in the amount of DNA were adjusted with the empty expression vector (pSG5). pCMV-gluc (0.3ng) was used as an internal control. Values are presented as the ratio of Fluc to Gaussia luciferase (Gluc) activities. Data are derived from at least three independent experiments performed in duplicate. Error bars indicate the standard errors of the means.
Fig 4.
HPV1 and 8 E8^E2C proteins interact with NCoR/SMRT components in an E8-dependent manner.
(A) 293T cells or HeLa cells were transfected with expression vectors for HA-tagged HPV1 and 8 E8^E2C wt or K2A d3-10 mutant proteins and whole cell lysates were directly analyzed (input) or precipitated with α-HA antibody (IP) and subjected to immunoblot analysis with the indicated antibodies. (B) E8 consensus sequences for alpha-PV (a1, 5, 6, 7, 8, 9, 10, 11 and 13 subgroups) and beta-PV (b1-6 subgroups) were obtained by extracting and aligning HPV E8^E2C sequences using the papillomavirus episteme database (http://pave.niaid.nih.gov [35]) and WebLoGo version 2.8.2 [36]. (C) The conserved KLK motif is important for the interaction of HPV8 E8^E2C with TBLR1 and HDAC3 and the transcription repression function. HeLa or RTS3b cells were transfected with expression vectors for HPV8 E8^E2C-HA wt or KLK mt—HA proteins and whole cell lysates were directly analyzed (input) or precipitated with α-HA antibody (IP) and subjected to immunoblot analysis with the indicated antibodies. (D) HeLa (left graph) or RTS3b (right graph) cells were transfected with the empty vector or HPV8 E8^E2C or E8^E2C KLK mt expression vectors (1ng), pC18-Sp1-luc (100ng) and pCMV-gluc (0.3ng). Luciferase activities were determined as described in Fig 3. Data are derived from three independent experiments and error bars indicate the SEM.
Fig 5.
The HPV16 and 31 E8^E2C proteins recruit the NCoR/SMRT complex to viral replication foci.
HeLa cells were transfected with 300ng of E1 expression vectors (pCMV neo 3xFlag-31E1 or pSG 3xFlag-16E1), 30 ng of E2 expression vectors (pSX 31 myc-E2 or pSG 16 E2) and 30 ng of the expression vectors for E8^E2C or the E8^E2C KWK mt proteins (pSG 31 E8^E2C HA/pSG 31 E8^E2C KWK mt HA or pSG 16 E8^E2C HA/pSG 16 E8^E2C KWK mt HA). Cells were stained with the indicated primary antibodies and analyzed by immunofluorescence microscopy. DNA was stained with DAPI (blue). Signals for E8^E2C (or the E8^E2C KWK mt) and NCoR/SMRT components in replication foci from 4–7 individual cells were analyzed for colocalization using optical sections and Zeiss Axiovision40 4.8.2 software. Colocalization is expressed as the Pearson´s correlation coefficient. Statistical significance between wt and mt was determined by Student´s t-test (**p<0.01; ***p<0.001).
Fig 6.
HPV 31 E8^E2C recruits NCoR/SMRT complexes to distinct nuclear foci.
RTS3b were transfected with a HPV31 URR reporter plasmid and expression vectors for E1 (500 ng) (pCMV neo 3xFlag-31E1), E2 (50 ng) (pSX 31 myc-E2) and E8^E2C or the E8^E2C KWK (50 ng) (pSG 31 E8^E2C HA or pSG 31 E8^E2C KWK mt HA). Cells were stained with (A) α-flag to detect E1 and α-HA to detect E8^E2C (KWK mt) proteins or (B) α-TBLR1 and α-HA to detect E8^E2C (KWK mt) proteins. Slides were then analyzed by immunofluorescence microscopy. DNA was stained with DAPI (blue). Signals for E8^E2C (or the E8^E2C KWK mt) and TBLR1 in replication foci from 7–9 individual cells were analyzed for colocalization using optical sections and Zeiss Axiovision40 4.8.2 software. Colocalization is expressed as the Pearson´s correlation coefficient. Statistical significance between wt and mt was determined by Students t-test (***p<0.001).
Fig 7.
HPV 8 E8^E2C recruits NCoR/SMRT complexes to distinct nuclear foci.
RTS3b were transfected with a URR reporter plasmid for HPV8 and with expression vectors for E1 (500 ng) (pCMV neo 3xFlag-8E1), E2 (50 ng) (pSG8 E2) and E8^E2C or the E8^E2C KLK mt proteins (50 ng) (pSG 8 E8^E2C HA or pSG 8 E8^E2C KLK mt HA). Cells were stained with the indicated primary antibodies and analyzed by immunofluorescence microscopy. DNA was stained with DAPI (blue). Signals for E8^E2C (or the E8^E2C KLK mt) and NCoR/SMRT complex components in foci from 6–9 individual cells were analyzed for colocalization using optical sections and Zeiss Axiovision40 4.8.2 software. Colocalization is expressed as Pearson´s correlation coefficient. Statistical significance between wt and mt was determined by Students t-test (**p<0.01; ***p<0.001).
Fig 8.
E8^E2C proteins inhibit replication in a reporter-based replication assay.
(A) Replication assay for HPV16 and HPV 31. HeLa cells were transiently transfected with a reporter plasmid containing the HPV16 or 31 URR and the viral early promoter driving the firefly luciferase gene (10 ng) (pGL 16 URR luc or pGL 31 URR luc) and expression vectors for E1 (100 ng) (pSG16 E1 or pSG31 E1), E2 (10 ng) (pSG16 E2 or pSX31 E2) and E8^E2C or the E8^E2C KWK mt (HPV16: 1 ng; HPV31: 10 ng) (pSG31 E8^E2C, pSG31 E8 KWK mt, pSG16 E8^E2C, pSG16 E8 KWK mt). (B) Replication assays for HPV1 and HPV8. RTS3b cells were transfected with the HPV1 reporter plasmid and expression vectors amounts described for HPV31 (pGL 1 URR luc, pSG 1 E1, pSG 1 E2, pSG 1 E8^E2C, pSG1 E8^E2C K2A d3-10 mt). RTS3b cells were transfected with the HPV8 reporter plasmid and expression vectors as described for HPV16 (pGL 8 URR luc, pSG 8 E1, pSG 8 E2, pSG 8 E8^E2C, pSG 8 E8^E2C K2A d3-10 mt). Differences in the amount of DNA were adjusted with the empty vector (pSG5). pCMV-gluc (0.3ng) was used as an internal control. Replication activity is shown as the relative replication measured by real-time PCR after DpnI-digestion (“relative replication (DpnI)”) (reporter plasmid cotransfected with the empty expression vector = “vector” is set to 1) or as the ratio between firefly luciferase (fluc) to gaussia luciferase (gluc) activities (“Fluc/Gluc”). Data are derived from at least three independent experiments performed in duplicate. Error bars indicate the standard errors of the means. A paired two tailed t-test was used to determine statistical significance (*p<0.05, **p<0.01).
Fig 9.
SiRNA combinations against HDAC3 and TBLR1 or NCoR and SMRT relieve the repression of replication mediated by E8^E2C proteins.
(A) HeLa cells were transfected with siRNA combinations (11.7pmol of each siRNA) against HDAC3 and TBLR1 or NCoR and SMRT and 24h later reporter and expression plasmids were transfected as described in Fig 7A. (B) RTS3b cells were treated with siRNA (15pmol of each siRNA) as described for A and transfected with plasmids as described in Fig 7B. Replication activity is shown as the relative replication measured by real-time PCR after DpnI-digestion (“relative replication (DpnI)”) (“E1/E2” is set to 1) or as relative replication with “E1/E2” set to 1 derived from the ratio of Fluc to Gluc activities. Data are derived from at least three independent experiments performed in duplicate. Error bars indicate the standard errors of the means. A paired two tailed t-test was used to determine statistical significance (*p<0.05, **p<0.01, ***p<0.001).
Fig 10.
SiRNA combination against NCoR and SMRT relieves the repression of transcription mediated by E8^E2C proteins.
(A) HeLa cells were transfected with siRNA combinations (11.7pmol of each siRNA) against HDAC3/TBLR1 or NCoR/SMRT and 24h later the cells were transfected with the pC18-Sp1-luc reporter (100 ng) and the expression vectors for HPV 16 or 31 E8^E2C (1 ng for HPV16, 10 ng for HPV 31) or the KWK mt proteins. (B) RTS3b cells were transfected with the siRNA combination (15pmol of each siRNA) against NCoR/SMRT and 24h later the cells were transfected with the pC18-Sp1-luc reporter (100 ng) and the expression vectors for HPV 1 or 8 E8^E2C (10 ng for HPV1, 0.2 ng for HPV 8) or the K2A d3-10 mt proteins. Differences in the amount of DNA were adjusted with the empty expression vector (pSG5). pCMV-gluc (0.3ng) was used as an internal control. Values are presented as the ratio of fluc to gluc luciferase activities. Data are derived from at least three independent experiments performed in duplicate. Error bars indicate the standard errors of the means. A paired two tailed t-test was used to determine statistical significance (*p<0.05, **p<0.01, ***p<0.001).
Fig 11.
Knock-down of NCoR/SMRT components by siRNA increases transcription of HPV31 genomes.
SCC13 cells were transfected with a control (siCon) or specific siRNAs (90pmol) and the next day with 1.3μg of re-ligated HPV31 wt (wt) or HPV31 E8 KWK mt (E8 KWK mt) genomes. RNA was isolated 48h later and analyzed by qPCR for the expression of spliced E1^E4, HDAC3, NCoR, SMRT, TBLR1 and PGK1 as a reference using specific primer pairs. Data are derived from four independent experiments and the values are presented relative to HPV31 wt/siControl. Error bars represent the SEM and statistical significance was tested by 1way ANOVA and Bonferroni´s multiple comparison test as a post test (*p<0.05).
Fig 12.
DN-NCOR activates viral transcription.
(A) RTS3b cells were transfected with the pSG-DN-NCoR plasmid and stained for DN-NCoR expression using an anti-Flag antibody by indirect immunofluorescence. DAPI was used to identify cell nuclei. (B) RTS3b cells were transfected with the reporter plasmid pC18-Sp1-luc (100ng) and expression vectors for HPV31 E8^E2C, E8^E2C KWK mt (10 ng) and DN-NCoR as indicated and pCMV-Gluc as a transfection control: Data are derived from three independent transfection experiments carried out in duplicate and are presented relative to pC18-Sp1-luc/empty vector. Error bars indicate the SEM. (C) SCC13 cells were transfected with 1.3μg each of HPV16 wt, HPV16 E8 KWK mt, HPV31 wt or HPV31 E8 KWK mt genomes and 0.5μg of the empty vector (vec) or pSG DN-NCoR plasmid. RNA was analyzed by qPCR using E1^E4 and PGK1 primers. Data are derived from five independent experiments and are presented relative to HPV wt/empty vector (vec). Error bars represent the SEM and statistical significance was tested by 1way ANOVA and Bonferroni´s multiple comparison test as a post test (*p<0.05; **p<0.01).
Fig 13.
DN-NCoR activates HPV31 genome replication.
SCC13 cells were co-transfected with 4μg religated HPV31 wt or E8 KWK mt genomes and 1.5μg of the empty vector pSG5 (-) or the pSG DN-NCoR plasmid. (A) Low-molecular weight DNA was isolated 72 p.t. and analyzed by Southern blotting after DpnI digestion to remove non-replicated genomes (non-rep.). M indicates 100 pg of a linearized HPV31 genome. (B) The graph shows data derived from four independent experiments which are presented relative to HPV wt/empty vector (vec). Error bars represent the SEM and statistical significance was tested by a paired Student´s t-test (*p<0.05).