A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter
- Chen Gao†1,
- Ming-Ming Pan†1, 2,
- Yan-Jun Lei1, 2,
- Li-Qing Tian3,
- Hui-Ying Jiang1,
- Xiao-Li Li1,
- Qi Shi1,
- Chan Tian1,
- Yu-Kang Yuan2,
- Gui-Xiang Fan2 and
- Xiao-Ping Dong1, 4Email author
© Gao et al; BioMed Central Ltd. 2012
Received: 31 August 2011
Accepted: 15 February 2012
Published: 15 February 2012
The human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells.
CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2.
These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.
KeywordsHPV-2 E2 DNA-binding Transcriptional regulation Promoter
Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect the mucosal epithelial tissues of anogenital tract, oral cavity and upper alimentary tract, as well as cutaneous epithelial tissues of hands, feet and trunks. HPVs have been grouped into cutaneous type that causes cutaneous warts and epidermodysplasia verruciformis, and mucosal type that predominantly induces benign and malignant lesions of the genital tract, in which HPV-2 has been frequently associated with verrucae vulgaris . HPV-2 genome is composed of eight open reading frames (ORFs) encoding the regulatory proteins essential for completion of the viral life cycle and the structural components of the virion, respectively .
HPVs' E2 proteins are believed to control the transcriptions of viral genes through binding to the specific sites in viral DNA, multiple copies of which are found in the viral upstream regulatory regions (URRs) . The HPV E2 protein can function as either a repressor or an activator of the early gene transcription, depending on the location of E2 binding sites in the viral regulatory region as previously demonstrated for genital HPVs . The structure of the E2 protein resembles a typical transcription factor, with an amino-terminal transcriptional activation domain (TAD) and a carboxyl-terminal DNA-binding/dimerization domain (DBD), separated by a variable hinge region . The E2 protein exists in solution and binds to the target DNA as a dimmer. The HPV-16 E2-DBD forms a dimeric β-barrel, with each subunit contributing an anti-parallel 4-stranded β-sheet "half-barrel" . Several studies showed that E2 acts as a transactivator at low concentrations, while as a repressor at high concentration. Recently, it has been reported that the locations of E2 binding sites are important for transcriptional repression, independent of binding affinities .
Besides being a transcriptional regulator in the life-cycle of virus, E2 protein is believed to play an important role in the carcinogenesis of HPV-associated cancers. The HPV genome can exist in the malignant cells in two forms, integrated into the host chromosome or episomal DNA. The majority of HPV-associated cancers, especially cervical carcinoma, contain integrate HPV DNA . Usually, integration of viral genome into host chromosome results in disruptions of E2 and E1 ORFs, leading to an increased transcription from the viral early promoter and elevated expression of viral oncogenes E6 and E7 [7, 8]. About 15-20% HPV-positive cervical cancers contain intact HPV genomes in extrachromosomal state. Various point mutations or deletions in the HPV genome were reported to be related to the viral oncogenesis potential, e.g. in the long control region (LCR) , E2 and E1 ORFs . Previously we reported a verrucae vulgaris patient with huge extensive clustered cutaneous who was confirmed to be infected by a HPV-2 variant [11, 12]. Several point mutations were detected in the LCR of this HPV-2 variant that lead to an increased promoter activity. In addition, five point mutations were found within the E2 ORF. Expression of the E2 mutant exhibited increased activities on the viral early promoter as compared with the prototype E2 .
In order to gain insight into the potential influences of these mutations within E2, we constructed a series of mammalian and prokaryotic expressing plasmids encoding E2 proteins with one to five amino acid (aa) substitutions. Upon co-transfection with a CAT reporter under the control of HPV-2 LCR, the E2 construct containing the A338V mutation within the DNA-binding domain functioned as a transactional activator instead of repressor. Electrophoretic mobility shift assays (EMSA) demonstrated that the ability of E2 protein with A338V mutation bind a double-stranded DNA sequence containing two E2 binding sites is markedly stronger than HPV-2 prototype E2. The binding affinity of the E2 A338V mutant for the promoter-proximal LCR sequences of HPV-2 and HPV-16 were also significantly increased. Structural analyses indicated that the mutation A338V located in the region of beta barrel. These results imply that the mutation A338V is critical for the E2 DNA-binding and promoter regulation.
The A338V mutation within the HPV-2 E2 DNA-binding domain is critical for E2 transcriptional regulation activity on the HPV-2 early promoter
Next, we examined the transcriptional activity of E2 with A338V mutation the HPV-negative cervical cancer cell lines C33A and the HPV-16 genome-containing SiHa cells. Consistent with the results in HeLa cells, under the control of HPV-2 LCR, co-expression of pcDNA-E2-proto and pcDNA-E2-Mut (-) led to obviously low CAT expressions compared with mock, whereas co-expressions of pcDNA-E2-Mut and pcDNA-E2-A338V caused high CAT expressions (Figure 2B). Notably, in pcDNA-E2-A338V-transfected cells, the relative CAT expression was higher than that of mock. These results imply that the transcriptional repression activity of E2 mutant A338V is independent of the endogenous HPV genome.
The A338V E2 mutant increased the binding capacity to the DNA sequences containing conservative E2 binding sites in vitro
In the context of E2 binding domain, both prototype E2 and the A338V mutant E2 formed protein-DNA complexes with the probes in a dose-dependent manner. Interestingly, the binding activity of E2 mutant was significant stronger than that of prototype E2 (Figure 4B). To confirm this phenomenon, four E2 proteins covering E2 hinge region and DNA-binding region were employed into EMSA. Figure 4C showed that the DNA-binding activity of E2-HC-A338V was stronger than that of prototype E2-HC. Additionally, E2-HC-Mut with A338V and other three point mutations in hinge region showed similar DNA-binding activity as E2-HC-338 V, while E2-HC-Mut (-) with only three mutations in hinge region caused similar DNA-binding activity as prototype E2-HC. As expected, neither GST nor E2 transactivation domain (E2-N), formed a complex in EMSA. Similar manner were observed in the EMSA in the context of full-length E2 protein, in which two constructs containing the A338V mutation (E2-FL-A338V and E2-FL-Mut) formed more obvious protein-DNA complexes than the other two constructs without the A338V mutation (E2-FL and E2-FL-Mut (-)), regardless of having the point mutations in the transactivation domain and hinge area (Figure 4D). These results highly suggest that the substitution of Ala to Val at residual 338 in HPV-2 E2 protein influences critically its DNA-binding affinity.
E2 DNA-binding affinities were influenced by the length of the E2 peptides
The A338V E2 mutants increased the binding affinity to the promoter-proximal LCR sequences of HPV-2 and HPV-16
To evaluate the DNA-binding activities of E2 with A338V to the wild-type HPV sequences, the biotin-labeled double-stranded oligos derived from the sequences of prototype HPV-2 and HPV-16 LCRs, which contained two E2 protein binding sites, were mixed with equal amount of the different recombinant prototype and mutated E2 proteins and assessed in EMSA. Consistent with results shown in Figure 4, the A338V E2 mutants showed clearly stronger binding affinities to both HPV-2 (Figure 5B) and HPV-16 (5 C) oligos than the HPV-2 prototype E2, in the context of either full-length or truncated forms. No difference was observed in the binding affinity of HPV-2 E2 to the LCR sequences of homologous or heterologous HPV genotypes. These results show that A338V E2 mutant has stronger binding affinity to the promoter-proximal LCR sequences of wild-type HPVs. E2 C-terminus (E2-C) possessed much stronger binding activities to HPV-2 and HPV-16 LCR than E2-HC and E2-FL, which were coincident well with the binding tendency of different E2 in length shown in Figure 4B, C and 4D. The multiple bands at higher molecular weight position in the gels (Figure 4C, D and 5) may represent the dimmers of the E2 proteins.
Co-expressions of HPV-2 E2 mutants with A338V induced more active activity on heterologous HPV-16 early promoter P97 than the HPV-2 prototype E2.
In this study we have provided evidence data that a naturally occurred mutation of A338V in HPV-2 E2 increases E2 DNA-binding capacity and reverses its transcriptional regulation activity on the viral early promoter. The effect of this mutation on the biological functions of E2 seems to be very critical, since the other four amino acid exchanges locate at the transactivation domain and the hinge regions of E2 have little impact. The E2 protein has the typical structure of transcriptional regulator, which consists of a multiple-protein-binding transactivation domain, a DNA-binding/dimerization domain, and a flexible linker . Consistent with other previous studies, our data confirm that although the C-terminal segment of E2 alone has DNA-binding capacity, lacking its N-terminal portion makes the truncated E2 almost loss it's of promoter repressor activity. Our data indicate that the E2 N-terminal alone works as a transcriptional activator, inducing about 1.5-fold increased promoter activity. However, this positive effect on the promoter is totally abolished in the context of whole E2 protein. The substitution of L118S in the E2 transactivation domain shows no influence on either DNA-binding or promoter activity. The contribution of E2 hinge region to its transcriptional regulator is believed to be not essential , as three naturally occurred mutations in this area together do not influence either the E2 DNA-binding or transcriptional activation.
Previous study has showed that binding of the full-length wild-type BPV-1 E2 protein to the LCR sequences leads to formation of DNA loops and the transcriptional activating domain of E2 is necessary for this loop . Such structure will result in the tissue-specific enhancers shifting closer to the core transcription complex for transcriptional activation [20, 21]. Meanwhile, some studies have indicated that binding of the intact E2 to the LCR sequences may spatially prevent the transcriptional machine to active the promoter, which are the main molecular mechanism for E2 transcriptional repression [22, 23].
E2 has also been shown to be able to interact with other cellular agents, e.g. Brd4, to regulate its transcriptional activity . The identification of Brd4 as a component in a dominant form of E2 complexes indicates that Brd4 may be the cofactor for HPV E2 repressor function . Apparently, Brd4 recruits E2 that in turn prevents the recruitment of TFIID and pol II to the HPV promoter . Amino acid substitutions within the E2 transactivation domain impaired both the transcriptional activity and binding to Brd4 . Furthermore, Brd4 is a host chromatin adaptor for papillomavirus. The dimerization of the E2 is required for efficient Brd4 binding . The mutation from Ala to Val at aa 338 of HPV-2 E2, which would change the hydrophobicity and/or tertiary structure of E2, will lead to a modification of its interaction with the chromatin, and thus, modulates its transcriptional regulation activity. Although our data highlight a close correlation between the increased activity for DNA-binding and the enhanced activity for viral early promoters of the mutated E2 protein, the exact mechanism remains unclear.
Our data indicate that the DNA-binding capacity of the C-terminal fragment of E2 is stronger than those with the hinge region, and much stronger than the full-length E2. Earlier study has found that besides the full-length E2, bovine papillomavirus (BPV) E2 ORF also encodes two other E2 peptides, E2-TR and E8/E2 proteins . These shorter E2 proteins contain the DNA binding and dimerization domains of the C-terminus and hinge region, but lack the transactivation domain. Relative abundances of the truncated E2 proteins have been observed in BPV transformed cells (the molar ratio of E2:E2-TR:E8/E2 is 1:10:3) . Expression of HPV-31 E8E2C protein has been reported to be able to inhibit HeLa cell growth . However, the transcriptional profiles of other HPV E2 ORFs, regardless in benign or malignant cells, are rarely addressed. The fact that the C-terminus E2 binds DNA stronger suggests that it is more competitive than the full-length E2 in the cells.
Our study provides the evidence that HPV-2 E2, regardless of wild-type or mutant (A338V), induces the similar biological effectiveness under the controls of the homologous and heterologous HPV LCRs. This suggests that E2 protein may induce same regulative activity on the viral early promoters from different HPVs with similar upstream components. Although there are more than 100 genotypes of HPVs involving in various human benign or malignant proliferating diseases, the sequences of viral genomes are relatively conservative. Hence, the effectiveness of HPV-2 E2 may represent a common property of HPVs' E2 proteins.
In addition to the role in regulating viral transcription, HPV E2 protein involves in enhancing E1-dependent viral DNA replication and genome maintenance. In HPV genomes the viral DNA replication initiation site co-localizes with the viral transcription region. However, the regulative function of E2 in viral DNA replication is far from understood compared with its role in transcriptional regulation. Although the point mutations in TAD and in hinge region within this E2 mutant do not affect DNA-binding and transcriptional regulation, their influence on viral genome replication cannot be excluded. Sequences analyses of this variant HPV-2 strain have also identified several point mutations in its E1 ORF. Further studies of viral genome replication will help explore the inconvenient reason of such huge verrucae vulgaris.
Our study provides evidence that HPV-2 E2 with Ala to Val mutation at aa 338 is critical for E2 DNA-binding and its transcriptional regulation. The binding abilities of E2 proteins with A338V to either an artificial probe containing with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of HPV-2 prototype E2. Furthermore, co-expression of the E2 constructs containing A338V mutation induces higher activities on heterologous HPV-16 early promoter P97 than that of prototype E2.
The primers for site-directed mutation PCR
The sequence of the primers
PCR amplification was performed in 25 μl of a reaction mixture containing 2.5 U of Taq DNA polymerase (TaKaRa, Dalian, China), 20 mM dNTP, 150 ng of each mixture of HPV-2 E2 specific primers at the cycle condition of denaturing at 94°C for 30 s, annealing at 58°C for 30 s, extending at 72°C for 1 min, totally 30 cycles, respectively. Briefly, to construct the E2-sequence containing single point-mutation including the mutants of L118S, S235P or A338V, two separated PCR amplifications were conducted using pcDNA-E2-proto as the templates, with the primer mixture of E2-up with 118-down, 235-down or m338-down, and the mixture of E2-down with 118-up, 235-up or m338-up, respectively. After purified, two individual PCR products were mixed and annealed, and the sequence covering whole E2 ORF of each mutant was constructed by another PCR amplification with primers E2-up and E2-down, generating E2-L118S, E2-S235P and E2-A338V, respectively. To generate E2-sequence containing two point-mutations of L118S and S235P, two separated PCRs were conducted based on the sequence of E2-L118S, with the primes E2-up and 235-down, as well as 235-up and E2-down, respectively. The whole E2 sequence of this mutant was obtained with the same protocol above, generating E2-L118S/S235P. To construct E2-sequence containing other four mutations except A338V, the PCR reactions were separately performed using E2-Mut sequence as the template, with the primer E2-up and 338-down, as well as 338-up and E2-down. The whole E2 sequence was obtained based on the protocol above, generating E2-Mut (-). E2 N-segment (from nt 2685 to 3279) and E2 C-segment (from nt 3618 to 3860) were generated by PCR with respective primer mixtures. The generated E2 sequences were cloned into plasmid pMD18-T. After verified with sequencing assays, various E2 segments were released from cloning vectors and subcloned into pcDNA3.1, generating mammalian expressing recombinant plasmids pcDNA-E2 (Figure 1A).
To construct the different HPV-2 E2 prokaryotic expressing plasmids, including E2 of prototype (E2), E2 of isolate 1 (E2-Mut) and various mutated E2, three lengths of E2 sequences, including the full-length E2 ORF (from aa 1 to 391, FL), the sequence starting from hinge region to the end (from aa 197 to 391, HC) and C-terminal segment (from aa 311 to 391, C), were generated by PCR technique with different primer mixtures, using individual pcDNA-E2 as the templates. The PCR products were cloned into plasmid pMD18-T and subcloned into a (GST) expression vector pGEX-2 T, generating various plasmids pGST-E2 (Figure 2A).
Cell line, transfection and CAT assay
The human cervical cancer cell lines HeLa were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal calf serum (HyClone). C33A and SiHa cell lines were maintained in ATCC-formulated Eagle's Minimum Essential Medium (Catalog No.30-2003) with 10% fetal calf serum. Cells were plated into 60 mm 6-well plates (Falcon, Japan) one day before transfection. 2 μg of plasmid pCAT-LCR were transfected with Lipofectamine 2000 transfection reagent (Invitrogen, USA), together with 1 μg of pCMV-β-galactosidase as internal control. To evaluate the effectiveness of E2 protein on the promoter activity, various E2 expression plasmids (500 ng) were co-transfected into cells. Cells were harvested at 48 h after transfection.
CAT expressions were measured quantitatively using a CAT ELISA kit (Roche, Switzerland), according to the instruction manual. The expression of β-galactosidase activity was determined using O-nitrophenyl-β-D-galactopyranoside (ONPG) as a colorimetric substrate. HPV-2 promoter activities were determined by calculating the rates of CAT and β-galactosidase values. Each experiment was independently performed for three to five times.
HeLa and C33A cells transfected with 500 ng various E2 expressing plasmids, together with or without 2 μg pCAT-LCR, were harvested 24, 48 and 72 h post-transfection. Cells were pelleted by short centrifugation and suspended in the lysis buffer (10 mM Tris-HCl, pH 7.8, 0.5% sodiumdeodycholate, 0.5% Nonidet P-40, 100 mM NaCl, 10 mM EDTA), supplemented with complete proteasomal inhibitor mixture. Cell lysates were separated by 15% SDS-PAGE and electro-transferred onto nitrocellulose membranes. After blocking with 5% nonfat-dried milk in PBS (phosphate buffered saline, pH 7.6) overnight at 4°C, the membranes were incubated with 1:1,000 HPV E2 specific monoclonal antibody at room temperature (RT). After washing with PBST (phosphate buffered saline, pH 7.6, containing 0.05% Tween-20), the membranes were incubated with 1:5,000 horseradish peroxidase (HRP)-conjugated anti-mouse antibody. The E2 protein signals were visualized by ECL kit (PE Applied Biosystems, USA). To reuse the blotted membrane, the developed membrane was treated in the Restore Western Blot Stripping Buffer (Thermo, USA) for 10 min at RT. 1:1,000 mAb anti-human β-actin (Santa Cruz, USA) and HRP-conjugated anti-mouse antibody were used to identify β-actin protein. ECL kit was used to visualize the signals.
Expression and purification of E2 proteins
The recombinant prokaryotic proteins tagged with GST were bacterially expressed in E. coli BL21 and purified with Glutathione Sepharose 4B Agarose (Pharmacia, USA) according the protocol described in our previous study . The purities of the purified proteins were verified by 15% SDS-PAGE.
Electrophoretic mobility shift assays (EMSA)
For EMSA, single DNA oligos labeled with biotin that covered two E2 binding sites (HPV-E2BS) ACCGAAAACGGTCAGACCGAATTCGGTTGT3' and 5'ACAACCgAATTCGGTCTGACCGTTTTCGGTCACAC3') and HPV-16 (5'GGCGTAACCGAAATCGGTTGAACCGAAACCGGTT3' and 5'AACCGGTTTCGGTTCAACCGATTTCGGTTACGCC3') were synthesized based on the sequences in GenBank. After denaturing by heating, two signal DNA oligos were annealed at RT to a double-stranded DNA probe. 12.5 to 250 fM biotin-labeled oligonucleotide probes were mixed with various amounts of different E2 proteins in 20 μl binding buffer (10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5) at RT for 20 min. For competition experiments, biotin-labeled oligo HPV-E2BS was competed with 50-, 100- and 500-fold excess of unlabeled homologous oligo or 500-fold excess of unlabeled heterologous oligo (T7 promoter double-stranded sequences). For supershift EMSA, 7 μg recombinant HPV-2 E2-FL was incubated with mAb against HPV-E2 at RT for 30 min, prior to mixing with biotin-labeled oligo HPV-E2BS. DNA-protein complexes were separated from unbound probe in a 6.5% non-denaturing polyacrylamide gel and visualized by LightShift® Chemiluminescent EMSA Kit (Pierce, USA) according to the manufacturer's instruction. Quantitative analysis of images was carried out using computer-assisted software Image Total Tech (Pharmacia, USA). The image was scanned with Typhoon (Pharmacia, USA), digitalized and saved as TIF format.
The 3D structures of prototype and mutated HPV-2 E2 DNA-binding domain were modeled based on the existed crystal structure of HPV E2 with the help of software Modeller9.5 and NAMD2.6 (optimize the structure by energy minimization). The quaternary structure of HPV-18 (PDB: 1F9F) was choose as the template.
Molecular models of prototype and mutated HPV-2 E2 DNA-binding domain were constructed using the homology modeling software Modeller v9.5 . The closely related structure of HPV-18 E2 DNA-binding domain (PDB: 1F9F)  was used as the template. The resulting structure files were subjected to energy minimization using NAMD2.6 .
The human papillomavirus
Electrophoresis mobility shift assays
Open reading frames
Long control region
Upstream regulatory regions
Transcriptional activation domain
E2 protein binding sites
This work was supported by SKLID development Grant (2008SKLID202 and 2008SKLID102), Chinese National Natural Science Foundation Grant (81100980, 81101302 and 31100117), National Basic Research Program of China (973 Program) (2007CB310505), China Mega-Project for Infectious Disease (2009ZX10004-101).
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