Article Figures & Data
Figures
- Fig. 1.
Growth-inhibitory effect of papillomavirus E1 expression on CV-1 cells. (A) Map of the E1 and E2 proteins. The E1 protein is tagged with a Glu-Glu (EE) epitope and is expressed from the pMEP9 vector. The E2 protein is tagged with a Flag epitope and is expressed from the pMEP4 vector. E1 has three major domains: the N-terminal domain (N), the origin binding domain (OBD), and a C-terminal helicase domain. The E2 protein has two domains: the transactivation (TA) and DNA binding (DBD) domains. In most cases, a part of or the entire gene was codon optimized for increased protein expression. Recoded amino acid residues were BPV1 E1 (1 to 274), BPVE2 (none), HPV8 E1 (1 to 239), HPV8 E2 (none), HPV11 E1 (1 to 649), HPV11E2 (1 to 148), HPV16 E1 (1 to 151), HPV16 E2 (1 to 227), HPV31 E1 (1 to 402), and HPV31 E2 (1 to 372). (B) Western blot analysis of the E1 and E2 proteins expressed in transiently transfected CV-1 cells. Cells were transfected with combinations of pMEP9-E1, pMEP4-E2, or the empty pMEP4 or pMEP9 plasmid. Cells were treated with 3 μM CdSO4 for the last 4 h prior to extraction at 48 h posttransfection. The blot was probed with anti-EE for E1 and anti-Flag for E2. The HPV11 E1 and HPV31 E2 and E1-E2 protein lysates were diluted 5-fold with lysate from nontransfected cells to reduce the visual interference of overexposure from these samples (marked with asterisks). A total of 6 μg of cell lysate was loaded in each lane. (C) Cell growth suppression assay in CV-1 cells. CV-1 cells were transfected with papillomavirus E1 and E2 expression plasmids balanced with the respective empty vectors, pMEP4 and pMEP9. Cell growth was compared to that of the positive control or pMEP4 or -9 vector and the negative-control vector pTZ18R. After 24 h, cells were split 1:9 into 100-mm-diameter plates. After approximately 2 weeks of selection in 200 μg/ml hygromycin- and 400 μg/ml G418-containing medium, cells were fixed with formalin and the resulting colonies were stained with methylene blue. Similar results were obtained with and without CdSO4 in the medium.
- Fig. 2.
HPV16 E1 activates the pChk1, pChk2, and γH2AX proteins. CV-1 cells were transfected with an HPV16 E1 expression plasmid or empty vectors (pMEP9) on coverslips. Cells were stained and with chicken anti-EE (α-E1) (in green) and antibodies to various DNA damage response (DDR) proteins, pChk1 (Ser345), pChk2 (Thr68), and histone γH2AX (Ser139) (in red).
- Fig. 3.
Nuclear expression of papillomavirus E1 proteins induces phosphorylated Chk2. (A) Increased levels of phosphorylated Chk2 are detected in HPV16 E1-expressing cells. CV-1 cells were transfected with HPV16 E1 and E2 expression vectors or the corresponding empty vectors. Cells were stained with chicken anti-EE (shown in green), M2 or rabbit anti-Flag (in cyan), and anti-phosphorylated Chk2 (Thr68) (in red.) The imaging procedure is as described in the legend for Fig. 2A except that a 40× oil immersion objective with zoom 1 was used instead of the 63× objective lens. (B) Quantitative representation of the level of phosphorylated Chk2 signal in the presence (+) or absence (−) of detectable nuclear E1 or E2 or E1-E2 together. IMARIS version 7.2 was used to analyze the level of phosphorylated Chk2 in each cell. Briefly, each individual nucleus was analyzed for the intensity of signals from four different color channels using a surface tool. Approximately 10 or more fields of panels containing 30 to 150 nuclei from each sample were counted (totaling 600 to 1,000 nuclei per sample). A background threshold was subtracted from each signal. Nuclei that were expressing E1 or E2 above threshold levels were scored as positive, while cells that had expression lower than the threshold were scored negative. (C) The E1 protein from several different papillomavirus types induces pChk2. CV-1 cells were transfected with HPV11, -31, or -8 or BPV1 E1 or E1-E2 expression vectors as described above for panel A. The images of E2 expression alone are not shown. Quantitative analyses as described for panel B are shown to the right of the panel of confocal images.
- Fig. 4.
HFKs containing replicating HPV genomes show upregulation of phosphorylated Chk2. Primary HFKs and HFKs immortalized by and containing replicating HPV16, -18, and -31 genomes were grown on coverslips and fixed in 4% PFA. The cells were stained with antibodies against phosphorylated Chk2 (Thr68) and processed as described in Materials and Methods.
- Fig. 5.
The alphapapillomavirus E1 and E2 proteins colocalize in defined nuclear foci. (A) HPV16 E1 and E2 nuclear foci. CV-1 cells cultured on slides were transfected with HPV16 E1 and E2 expression plasmids or the corresponding empty vectors. The E1 and E2 proteins were detected with chicken anti-EE (α-E1) and mouse M2 anti-Flag (α-E2) antibodies, respectively, and DyLight secondary antibodies. Cellular DNA was counterstained with DAPI (blue). The E1 protein is detected as green and E2 as red. (B) HPV11 and -31 E1 and E2 nuclear foci. CV-1 cells were transfected with HPV11 or -31 E1 and E2 expression vectors as described for panel A. The characteristic foci seen with E1 and E2 coexpression are not observed when the proteins are expressed individually, similar to the observation with HPV16 (data not shown). (C) HPV16 E1 and E2 coexpressed in C33A and HFK localize to defined foci. HFK and C33A cells were transiently transfected with HPV16 E1 and E2 expression plasmids. For HFK, the cells were fixed with 4% PFA 24 h after transfection with 3 μM CdSO4 induction 4 h prior to fixation. The staining and imaging procedures are described in Materials and Methods.
- Fig. 6.
HPV16 E1 and E2 foci partially colocalize with DDR proteins from the ATM pathway. HFKs conditionally immortalized by culture in Y-27632 were transfected with HPV16 E1 and E2 expression plasmids or empty vectors (pMEP4/9) on coverslips. Cells were stained and processed as described in Materials and Methods, with chicken anti-EE (α-E1) (in green), M2 or rabbit anti-Flag (α-E2) (in cyan), and various DNA damage response (DDR) proteins, anti-phosphorylated ATM (Ser1981), p53 (Ser15), and histone γH2AX (Ser139) (in red).
- Fig. 7.
The E1-E2 foci recruit DDR proteins by causing cellular genomic instability. (A) EdU incorporation in E1-expressing cells. HFKs conditionally immortalized by culture in Y-27632 were transfected with HPV16 E1, E2 (total DNA amount balanced with empty vectors), E1-E2, and pMEP4/9 expression vectors. Cells were treated with 3 μM CdSO4 for 4 h prior to fixation and 50 μM EdU for 30 min prior to fixation. The procedure provided by the Click-iT EdU cell proliferation assay was used for the detection of EdU (shown in cyan) followed by immunostaining of E1 (α-E1; in green) and E2 (α-E2; in red) as described in Materials and Methods. (B) Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) signal incorporation in E1-expressing cells. HFKs conditionally immortalized by culture in Y-27632 were transfected with HPV16 E1, E2 (total DNA amount balanced with empty vectors), E1-E2, and pMEP4/9 vectors. Cells were treated with 3 μM CdSO4 4 h prior to fixation at 24 h posttransfection. Cells on coverslips were fixed in 4% PFA, permeabilized in 0.1% Triton X-100, and subjected to the TUNEL reaction. The cells were subsequently stained with anti-EE (α-E1) and anti-Flag (α-E2) antibodies, as described in Materials and Methods. The observed nonnuclear speckles in the TUNEL channel are due to transfected DNA.
- Fig. 8.
(A) HFKs conditionally immortalized by culture in Y-27632 were transfected with HPV16 E1 and E2 expression plasmids along with either an HPV16 origin-containing plasmid or HPV16 genomes (either prototype or W12) or a nonspecific plasmid, pTZ19U (control). Cells were stained with chicken anti-EE (α-E1; in green), rabbit anti-Flag (α-E2; in cyan), and anti-NBS1 (in red). (B) For each group of transfected cells, those displaying E1-E2 foci were divided into the three categories shown, according to the size of the foci. Between 38 and 54 cells were counted in each category. The percentage of cells in each category is shown.
- Fig. 9.
Nuclear focus formation requires the specific DNA binding and ATPase functions of E1 and the transactivation and E1 interaction functions of the E2 protein. (A) Diagram of the specific mutations generated in the HPV16 E1 and E2 proteins. The following mutations were made: E1 mutations that eliminate specific DNA binding (DB-) and ATP binding (ATP-) activities; mutations in E2 that interfere with interaction with the E1 protein (Int-), specific DNA binding (DB-), and the transcriptional regulation function of E2 (TA-). (B) Western blotting of mutated E1 and E2 proteins transiently expressed in CV-1 cells. CV-1 cells were transfected with E1 or E2 expression plasmids and corresponding empty vectors. Cells were treated with 3 μM CdSO4 4 h prior to harvesting at 48 h posttransfection. Proteins were detected with anti-EE for E1 (α-E1) and anti-Flag for E2 (α-E2). (C) The characteristic foci observed with wild-type E1 and E2 require the ATPase and DNA binding functions of E1 and the transactivation and E1 interaction functions of E2. CV-1 cells were transfected with wild-type and mutated E1 or E2 expression vectors as described for panel A, and cells were stained with antibodies against the EE epitope (α-E1), Flag (α-E2), and γH2AX (α-γH2AX). The staining and imaging procedures and cell growth suppression assay are as described in Materials and Methods.
Tables
- Table 1.
Activities of the E1 and E2 proteins required for ATM DNA damage response, growth suppression, and nuclear focus formation
Phenotype Protein expresseda None E1 wt E1 DB− E1 ATP− E1 wt + E2 wt E1 DB− + E2 wt E1 ATP− + E2 wt E1 wt + E2 Int− E1 wt + E2 TA− E1 wt + E2 DB− ATM induction − +++ − − +++ − − +++ +++ +++ Growth suppression − ++ − − +++ − − +++ +++ +++ Formation of E1/E2 DDR foci − − − − +++ − − − − +++ ↵a wt, wild type. Mutation effects are described as follows: DB− and ATP−, elimination of specific DNA binding and ATP binding activities, respectively; Int− and TA−, interference with interaction with the E1 protein and the transcriptional regulation function of E2, respectively. The relative magnitude of each activity is indicated as follows: +++, high; ++, medium; −, none.
