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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.

Gao C, Pan MM, Lei YJ, Tian LQ, Jiang HY, Li XL, Shi Q, Tian C, Yuan YK, Fan GX, Dong XP - BMC Mol. Biol. (2012)

Bottom Line: 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.These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changbai Rd 155, Beijing 102206, People's Republic of China.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

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The influences of various HPV-2 E2 constructs on the promoter activity under control of the LCR of HPV-2. A. The relative CAT expressions co-transfected with different E2 expressing plasmids in HeLa cells. The schematic structure of HPV-2-LCR cloned in the CAT reporter plasmid pBL-CAT6 is shown above. HPV-2-LCR covers the sequences from nt 6934 to 134. The positions of TATA box and four potential E2 binding sites are indicated with the starting nucleotide (nt) below. HeLa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as internal control. Cells were harvested 48 h after transfection. The expressions of CAT and β-galactosidase were determined. The CAT expression each preparation was normalized with its β-galactosidase value. The relative CAT expressions are averaged from at least three independent experiments and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM. B. The relative CAT expressions co-transfected with different E2 expressing plasmids in C33A and SiHa cells. C33A and SiHa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as an internal control. The relative CAT expressions are averaged from at least three independent experiments in C33A and SiHa cells and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM
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Figure 2: The influences of various HPV-2 E2 constructs on the promoter activity under control of the LCR of HPV-2. A. The relative CAT expressions co-transfected with different E2 expressing plasmids in HeLa cells. The schematic structure of HPV-2-LCR cloned in the CAT reporter plasmid pBL-CAT6 is shown above. HPV-2-LCR covers the sequences from nt 6934 to 134. The positions of TATA box and four potential E2 binding sites are indicated with the starting nucleotide (nt) below. HeLa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as internal control. Cells were harvested 48 h after transfection. The expressions of CAT and β-galactosidase were determined. The CAT expression each preparation was normalized with its β-galactosidase value. The relative CAT expressions are averaged from at least three independent experiments and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM. B. The relative CAT expressions co-transfected with different E2 expressing plasmids in C33A and SiHa cells. C33A and SiHa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as an internal control. The relative CAT expressions are averaged from at least three independent experiments in C33A and SiHa cells and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM

Mentions: Under our experimental condition, transfection of the blank CAT reporter vector (pBL-CAT6) did not induce detectable CAT expression (data not shown). Consistent with previous observations in HeLa cells containing HPV-18 genome [13], co-transfection of pcDNA-E2-proto significantly reduced the HPV-2 LCR driven CAT expression (Figure 2A, pcDNA-E2-proto), whereas co-transfection of pcDNA-E2-Mut significantly increased the CAT expression (pcDNA-E2-Mut). Co-transfected with the plasmids encoding N- (pcDNA-E2-N) and C-terminal (pcDNA-E2-C) E2 resulted in significantly higher CAT expressions than that with the plasmid encoding full-length prototype E2. Transfection of the plasmids expressing single point mutation within the transactivation domain (Figure 2A, pcDNA-E2-L118S) and the hinge region (Figure 2A, pcDNA-E2-S235P) resulted in comparable CAT expressions as that of prototype E2, while pcDNA-E2-L118S/S235P induced a relatively higher CAT expression. Interestingly, transient transfection of pcDNA-E2-A338V containing a single point mutation in the E2 DNA-binding domain led to a significantly increased CAT expression that was even slightly higher than that of pcDNA-E2-Mut. In contrast, transfection of pcDNA-E2-Mut (-) containing all four point mutations but A338V caused a significant repression of CAT expression that was comparable with pcDNA-E2-proto. These results demonstrate that the A338V mutation within the DNA-binding domain is essential for the E2 repression activity.


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.

Gao C, Pan MM, Lei YJ, Tian LQ, Jiang HY, Li XL, Shi Q, Tian C, Yuan YK, Fan GX, Dong XP - BMC Mol. Biol. (2012)

The influences of various HPV-2 E2 constructs on the promoter activity under control of the LCR of HPV-2. A. The relative CAT expressions co-transfected with different E2 expressing plasmids in HeLa cells. The schematic structure of HPV-2-LCR cloned in the CAT reporter plasmid pBL-CAT6 is shown above. HPV-2-LCR covers the sequences from nt 6934 to 134. The positions of TATA box and four potential E2 binding sites are indicated with the starting nucleotide (nt) below. HeLa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as internal control. Cells were harvested 48 h after transfection. The expressions of CAT and β-galactosidase were determined. The CAT expression each preparation was normalized with its β-galactosidase value. The relative CAT expressions are averaged from at least three independent experiments and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM. B. The relative CAT expressions co-transfected with different E2 expressing plasmids in C33A and SiHa cells. C33A and SiHa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as an internal control. The relative CAT expressions are averaged from at least three independent experiments in C33A and SiHa cells and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: The influences of various HPV-2 E2 constructs on the promoter activity under control of the LCR of HPV-2. A. The relative CAT expressions co-transfected with different E2 expressing plasmids in HeLa cells. The schematic structure of HPV-2-LCR cloned in the CAT reporter plasmid pBL-CAT6 is shown above. HPV-2-LCR covers the sequences from nt 6934 to 134. The positions of TATA box and four potential E2 binding sites are indicated with the starting nucleotide (nt) below. HeLa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as internal control. Cells were harvested 48 h after transfection. The expressions of CAT and β-galactosidase were determined. The CAT expression each preparation was normalized with its β-galactosidase value. The relative CAT expressions are averaged from at least three independent experiments and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM. B. The relative CAT expressions co-transfected with different E2 expressing plasmids in C33A and SiHa cells. C33A and SiHa cells were transfected with 2 μg of plasmid pCAT-LCR and 500 ng of either mock plasmid (pcDNA3.1) or the plasmids for various E2 constructs (as indicated). 1 μg pCMV-β-galactosidase was transfected as an internal control. The relative CAT expressions are averaged from at least three independent experiments in C33A and SiHa cells and presented relative to that of pcDNA3.1. Data are represented as mean ± SEM
Mentions: Under our experimental condition, transfection of the blank CAT reporter vector (pBL-CAT6) did not induce detectable CAT expression (data not shown). Consistent with previous observations in HeLa cells containing HPV-18 genome [13], co-transfection of pcDNA-E2-proto significantly reduced the HPV-2 LCR driven CAT expression (Figure 2A, pcDNA-E2-proto), whereas co-transfection of pcDNA-E2-Mut significantly increased the CAT expression (pcDNA-E2-Mut). Co-transfected with the plasmids encoding N- (pcDNA-E2-N) and C-terminal (pcDNA-E2-C) E2 resulted in significantly higher CAT expressions than that with the plasmid encoding full-length prototype E2. Transfection of the plasmids expressing single point mutation within the transactivation domain (Figure 2A, pcDNA-E2-L118S) and the hinge region (Figure 2A, pcDNA-E2-S235P) resulted in comparable CAT expressions as that of prototype E2, while pcDNA-E2-L118S/S235P induced a relatively higher CAT expression. Interestingly, transient transfection of pcDNA-E2-A338V containing a single point mutation in the E2 DNA-binding domain led to a significantly increased CAT expression that was even slightly higher than that of pcDNA-E2-Mut. In contrast, transfection of pcDNA-E2-Mut (-) containing all four point mutations but A338V caused a significant repression of CAT expression that was comparable with pcDNA-E2-proto. These results demonstrate that the A338V mutation within the DNA-binding domain is essential for the E2 repression activity.

Bottom Line: 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.These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changbai Rd 155, Beijing 102206, People's Republic of China.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

Show MeSH
Related in: MedlinePlus