四川地区宫颈癌组织高危型人乳头瘤病毒E2基因突变和适应性进化分析
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摘要
目的：宫颈癌是世界范围内女性第二常见的恶性肿瘤。持续高危型人乳头瘤病毒感染是导致宫颈癌的主要原因。HPV E2蛋白由N端转录活化结构域、C端DNA结合域以及可变的铰链区组成,在病毒的转录和复制调节中起着重要的作用。HPV E2基因的突变和整合与HPV致癌机制有关。HPVE2蛋白能抑制E6、E7蛋白表达,HPV整合常在E2基因处发生断裂缺失,而E2基因的缺失导致E6、E7蛋白的过表达。调查我国四川地区宫颈癌组织中高危型HPV E2基因的结构和适应性进化特点,旨在为探讨其与宫颈癌发生的关系以及HPV E2蛋白在致癌机制中所起作用的进一步研究提供理论基础。方法：经病理证实的宫颈癌组织来自2008年至2009年间206例四川宫颈癌患者。先用DNeasy Tissue Kit (Qiagen)提取宫颈组织样品基因组,提取的基因组经过β-globin球蛋白验证。再用MY09/11和E6 E7特异性引物对DNA样品进行分型。并对各型阳性样品采用巢式PCR技术用HPVE2引物对各型HPVE2基因进行扩增。PCR产物经纯化后进行双向测序,以获得可靠的序列信息。所获得序列通过Vector NTI 6软件与Genebank所收录的原型序列进行比较,并确定和分析突变情况。HPV序列数据与其原型参考序列通过MrBayes v3.1.2和Mega4.0软件的最大简约法和邻接法创建进化树。然后,我们运用最大拟然法研究HPVE2蛋白的氨基酸位点所经历的正选择压力。并通SWISS-PDBViewer软件对HPVE2蛋白的高级结构进行预测。结果：与Genbank标准株对比,四川地区的高危型HPV各型HPVE2基因均发现断裂整合以及多处突变。HPV16、18、31、33、45、52、58和HPV59核苷酸的突变分别为1.82%、1.28%、0.09%、1.04%、1.26%、1.45%、0.65%和1.53%；而氨基酸突变分别为4.93%、2.19%、0.27%、1.98%、1.63%、2.99%、0.84%和3.51%。结果表明,HPV16、HPV18、HPV33、和HPV59的E2基因区错义/同义替换比率(dN/dS)超过1,而HPV31、HPV45、HPV52和HPV58E2基因没有任何位点经历正选择。同时,我们还发现很多突变位于E2蛋白的T细胞或B细胞抗原决定簇上。结论：我们的实验证明HPVE2基因常出现断裂整合,各型HPV均存在多处突变。尽管绝大多数位点受到净化选择压力作用或者处于中性进化,仍有数据表明HPV16、HPV18、HPV33、和HPV59的E2基因区经历了正性的达尔文选择。那些经历正选择作用的位点和突变位于HPVE2蛋白不同的序列功能区内,提示这些位点可能对相关的功能贡献决定性作用。特别是那些位于抗原决定簇得突变可能影响病毒的感染效率和病毒的免疫原性。本研究的结果为理解HPVE2蛋白的结构和功能、病毒进化、致癌机制以及免疫研究提供了一定的基础数据和参考依据。
bjective:High-risk Human papillomaviruses (HR-HPV) are associated with cervical cancer,which is the second most common cancer in women worldwide. Clinical, molecular, and epidemiological investigations have identified persistent infection with HR-HPV as the major cause of cervical cancer. It is composed of a C-terminal DNA-binding domain, an N-terminal transactivation domain and a flexible hinge. HPV E2 protein plays key roles in the regulation of viral gene transcription and DNA replication.The E2 protein can represse the transcription of the oncogenes E6 and E7. Mutation and integration of the E2 protein is associated with carcinogenesis of HPV. The genome integration of HPV usually disrupts E2 gene and causes an absence of E2 gene sequences. The lack of E2 gene could lead to the upregulated expression of E6 and E7 protein. To study the features of mutations and adaptive evolution of E2 genens of high-risk Human Papillomavirus in patients with cervical cancer in Sichuan area of China.Methods:Histolo-gically confirmed cervical cancer tissues were obtained from 206 patients in Sichuan area between 2008 and 2009. DNA was extracted from frozen tissue specimens by DNeasy Tissue Kit (QIAGEN) and the quality of extracted DNA was checked by PCR amplification of B-globin gene HPVgenotype was detected by a combination of MY09/11 consensus primers PCR and type-specific E6 E7 gene nested PCR The fragments of E2 genes of high-risk HPV including HPV16, HPV18, HPV31, HPV33, HPV45,HPV52,HPV58,and HPV59 were amplified by nested PCR. All positive PCR products of nested PCR were sequenced after they were purified. The mutations were analyzed and determined by Vector NTI 6 software.The HPVs prototype from GenBank was used as reference. Moreover,the phylogenetic trees were constructed by the minimum-evolution and neighbor-joining algorithm using MrBayes v3.1.2 and Mega software version 4.0.To investigate the molecular basis of HPVs evolution, Phylogenetic analysis by maximum likelihood were used to identify lineages and amino acid sites under selective pressure.Finally,we predict the higher structure of the HPV E2 proteins and energy with SWISS-PDBViewer software. Results:In some specimens of all type of HPVs, the complete or part HPVE2 gene failed to be amplified, suggesting that the HPV DNAs in those specimens lost some region of viral E2 gene and exist in the integrated form.The point mutation of the E2 genes of high risk were found in all of samples.Compared with these prototypes published in Genbank,there were several new point mutations in the E2 genes of high-risk HPV in Sichuan area except for HPV31 which had only one. Nucleotide variation within HPV16,18,31,33,45,52,58 and HPV59 was 1.82%、1.28%、0.09%、1.04%、1.26%、1.45%、0.65% and 1.53%, respectively, and amino acid variation was 4.93%,2.19%,0.27%,1.98%,1.63%,2.99%,0.84%,and 3.51%,respectively. Our results showed that10,1,1and58 codon sites in the E2 open reading frames (ORFs) of HPV16,HPV18, HPV33, and HPV59 had nonsynonymous/synonymous substitution rate ratios (dN/dS) over 1,however, no specific codons in E2 ORF of HPV31,HPV45,HPV52 and HPV58were under positive selection.Conclusion:These data indicate that the genome integration of HPV usually disrupts E2 gene and causes an absence of E2 gene sequences.There are also some mutations in the E2 ORFs. Although most sites were under purifying selection or neutral evolution, these data indicative the E2 ORFs of HPV16,HPV18, HPV33, and HPV59 were under positive Darwinian selection. In addition, no position in the E2 genes of HPV31,HPV45,HPV52,and HPV58 isolated from Sichuan area of China was under positive selection. Since these positive sites and mutations were located in different functional sequence segments,it may conclude that these sites are crucial to related function.Especially, the substantial E2 amino acid variation located in surface epitopes could affect efficiency of infection or alter viral immunogenicity. It is reasonable to antieipate that this study would provide potential values to understand the structure and function of HPV E2 proteins and a basis of data and a reference for future studies on cervical carcinogenesis,the evaluation of viruses and immunity.
bjective:High-risk Human papillomaviruses (HR-HPV) are associated with cervical cancer,which is the second most common cancer in women worldwide. Clinical, molecular, and epidemiological investigations have identified persistent infection with HR-HPV as the major cause of cervical cancer. It is composed of a C-terminal DNA-binding domain, an N-terminal transactivation domain and a flexible hinge. HPV E2 protein plays key roles in the regulation of viral gene transcription and DNA replication.The E2 protein can represse the transcription of the oncogenes E6 and E7. Mutation and integration of the E2 protein is associated with carcinogenesis of HPV. The genome integration of HPV usually disrupts E2 gene and causes an absence of E2 gene sequences. The lack of E2 gene could lead to the upregulated expression of E6 and E7 protein. To study the features of mutations and adaptive evolution of E2 genens of high-risk Human Papillomavirus in patients with cervical cancer in Sichuan area of China.Methods:Histolo-gically confirmed cervical cancer tissues were obtained from 206 patients in Sichuan area between 2008 and 2009. DNA was extracted from frozen tissue specimens by DNeasy Tissue Kit (QIAGEN) and the quality of extracted DNA was checked by PCR amplification of B-globin gene HPVgenotype was detected by a combination of MY09/11 consensus primers PCR and type-specific E6 E7 gene nested PCR The fragments of E2 genes of high-risk HPV including HPV16, HPV18, HPV31, HPV33, HPV45,HPV52,HPV58,and HPV59 were amplified by nested PCR. All positive PCR products of nested PCR were sequenced after they were purified. The mutations were analyzed and determined by Vector NTI 6 software.The HPVs prototype from GenBank was used as reference. Moreover,the phylogenetic trees were constructed by the minimum-evolution and neighbor-joining algorithm using MrBayes v3.1.2 and Mega software version 4.0.To investigate the molecular basis of HPVs evolution, Phylogenetic analysis by maximum likelihood were used to identify lineages and amino acid sites under selective pressure.Finally,we predict the higher structure of the HPV E2 proteins and energy with SWISS-PDBViewer software. Results:In some specimens of all type of HPVs, the complete or part HPVE2 gene failed to be amplified, suggesting that the HPV DNAs in those specimens lost some region of viral E2 gene and exist in the integrated form.The point mutation of the E2 genes of high risk were found in all of samples.Compared with these prototypes published in Genbank,there were several new point mutations in the E2 genes of high-risk HPV in Sichuan area except for HPV31 which had only one. Nucleotide variation within HPV16,18,31,33,45,52,58 and HPV59 was 1.82%、1.28%、0.09%、1.04%、1.26%、1.45%、0.65% and 1.53%, respectively, and amino acid variation was 4.93%,2.19%,0.27%,1.98%,1.63%,2.99%,0.84%,and 3.51%,respectively. Our results showed that10,1,1and58 codon sites in the E2 open reading frames (ORFs) of HPV16,HPV18, HPV33, and HPV59 had nonsynonymous/synonymous substitution rate ratios (dN/dS) over 1,however, no specific codons in E2 ORF of HPV31,HPV45,HPV52 and HPV58were under positive selection.Conclusion:These data indicate that the genome integration of HPV usually disrupts E2 gene and causes an absence of E2 gene sequences.There are also some mutations in the E2 ORFs. Although most sites were under purifying selection or neutral evolution, these data indicative the E2 ORFs of HPV16,HPV18, HPV33, and HPV59 were under positive Darwinian selection. In addition, no position in the E2 genes of HPV31,HPV45,HPV52,and HPV58 isolated from Sichuan area of China was under positive selection. Since these positive sites and mutations were located in different functional sequence segments,it may conclude that these sites are crucial to related function.Especially, the substantial E2 amino acid variation located in surface epitopes could affect efficiency of infection or alter viral immunogenicity. It is reasonable to antieipate that this study would provide potential values to understand the structure and function of HPV E2 proteins and a basis of data and a reference for future studies on cervical carcinogenesis,the evaluation of viruses and immunity.
引文
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1.Zigui Chen,Rob DeSalle,Mark Schiffman,et al.Evolutionary Dynamics of Variant Genomes of Human Papillomavirus Types 18,45,and 97.JOURNAL OF VIROLOGY,2009,83(3):1443-1455.
2. Daraporn Pittayakhajonwut,Peter C. Angeletti et al.Analysis of cis-elements that facilitate extrachromosomal persistence of human papillomavirus genomes [J]. Virology,2008;374:304-314
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4. Kristin Klucevsek, Mary Wertz, John Lucchi, et al.Characterization of the nuclear localization signal of high risk HPV16 E2 protein Virology[J],2007;360:191-198
5.Michael K. Baxter,Maria G. McPhillips, et al. The Mitotic Chromosome Binding Activity of the Papillomavirus E2 Protein Correlates with Interaction with the Cellular Chromosomal Protein, Brd4 JOURNAL OF VIROLOGY,2005;79(8):4806-4818
6.Kruppel U,Muller Schiffmann A, et al. E2 and the co-activator p300 can cooperate in activation of the human papillomavirus type 16 early promoter. Virology,2008;377:151-159
7.Gagnon D,Joubert S,Senechal H, et al. Proteasomal degradation of the papillomavirus E2 protein is inhibited by overexpression of bromodo-main-containing protein 4[J]. J Virol,2009;May,83(9):4127-4139
8. Francoise Thierry.Transcriptional regulation of the papillomavirus oncogenes by cellular and viral transcription factors in cervical carcinoma[J]. Virology,2009;384:375-379
9. Green KL,Gaston K.Development of a topical protein therapeutic for human papillomavirus and associated cancers[J].BioDrugs,2006; 20 (4):209-218
10. Guilherme Menegon Giesel, Lui's Mauri'cio T.R. Limac, Joana Faber-Baratac, Characterization of the papillomavirus a1E2 peptide unfolded to folded transition upon DNA binding [J]. FEBS Letters,2008;582:3619-3624
11. Mariano Dellarole,Ignacio E. Sanchez,Eleonora Freire, et al.Increased Stability and DNA Site Discrimination of "Single Chain" Variants of the Dimeric B-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator[J]. Biochemistry, 2007;46 (43):12441-12450
12. Faber Barata,J,Mohana Borges R, Lima LM. Specificity in DNA recognition by a peptide from papillomavirus E2 protein[J].FEBS Letters,2006;580:1919-1924
13.Guilherme Menegon Giesel, Lui's Mauri'cio T.R. Lima, Joana Faber-Barata,Characterization of the papillomavirus a1E2 peptide unfolded to folded transition upon DNA binding[J], FEBS Letters 582 (2008) 3619-3624.
14. Meelis Kadaja, Helen Isok-Paas, Triin Laos, et al. Mechanism of Genomic Instability in Cells Infected with the High-Risk Human Papillomaviruses, PLoS Pathog.2009 April; 5(4):1-16
15.Herdman MT, Pett MR,Roberts I;et al. Interferon-beta treatment of cervical keratinocytes naturally infected with human papillomavirus 16 episomes promotes rapid reduction in episome numbers and emergence of latent integrants.Carcinogenesis [J].2006,Nov;27(11): 2341-2353.
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17. Monica Cricca, Simona Venturoli, Elisa Leo, [J]. Disruption of HPV16 E1 and E2 genes in precancerous cervical lesions.J Virol Methods,2009;158:180-183
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21. Daraporn Pittayakhajonwut, Peter C. Angeletti.Amino acid substitutions that specifically impair the transcriptional activity of papillomavirus E2 affect binding to the long isoform of Brd4[J]. Virology,2007;358:10-17
35. Peng-Sheng Zheng, Jane Brokaw, et al. Conditional Mutations in the Mitotic Chromosome Binding Function of the Bovine Papillomavirus Type 1 E2 Protein[J]. JOURNAL OF VIROLOGY,2005;79(3): 1500-1509
22. Elena E. Hernandez-Ramon,Julie E. Burns,Wenke Zhang, et al.Dimerization of the Human Papillomavirus Type 16 E2 N Terminus Results in DNA Looping within the Upstream Regulatory Region[J].JOURNAL OF VIROLOGY,2008;May:4853-4861
23 Emiko Soeda, Maureen C. Ferran, Carl C. Baker, et al. Repression of HPV16 early region transcription by the E2 protein[J]. Virology,2006; 351:29-41
24. Peng-Sheng Zheng, Jane Brokaw, et al. Conditional Mutations in the Mitotic Chromosome Binding Function of the Bovine Papillomavirus Type 1 E2 Protein[J]. JOURNAL OF VIROLOGY,2005;79(3): 1500-1509
25. Ciaran B. J. Woodman, et al.The natural history of cervical HPV infection:unresolved issues [J]. NATURE,2007;7:11-22.
[2]Zigui Chen,Rob DeSalle,Mark Schiffman,et al.Evolutionary dynamics of variant genomes of human papillomavirus types 18,45,and 97[J].J Virol,2009,83(3):1443-1455.
[3]Daraporn Pittayakhajonwut,Peter C.Angeletti.Analysis of cis-elements that facilitate extrachromosomal persistence of human papillomavirus genomes [J].Virology,2008,374(2):304-314.
[4]Song H, Moseley PL, Lowe SL,et al.Inducible heat shock protein 70 enhances HPV31 viral genome replication and virion production during the differentiation-dependent life cycle in human keratinocytes [J].Virus Res,2010,147(1):113-122.
[5]Gammoh N,Isaacson E,Tomaic V,et al.Inhibition of HPV-16 E7oncogenic activity by HPV-16 E2[J].Oncogene.2009,28(23): 2299-2304.
[6]Kruppel U,Muller Schiffmann A, Baldus,SE,et al. E2 and the co-activator p300 can cooperate in activation of the human papillomavirus type 16 early promoter [J]. Virology,2008,377(1): 151-159.
[7]Guilherme Menegon Giesel,Lui's Mauri cio T.R. Lima Joana Faber-Barata.Characterization of the papillomavirus a1E2 peptide unfolded to folded transition upon DNA binding [J].FEBS Letters,2008,582:3619-3624.
[8]Blakaj DM,Fernandez-Fuentes N,Chen Z,et al.Evolutionary and biophysical relationships among the papillomavirus E2 proteins [J].Front Biosci,2009,1(14):900-917.
[9]Dellarole M, Sanchez IE, Freire E,et al.Increased stability and DNA site discrimination of "single chain"variants of the dimeric β-barrel DNA binding domain of the human papillomavirus E2 transcriptional regulator [J].Biochemistry,2007,46(43):12441-1245O.
[10].Faber Barata,J,Mohana Borges R, Lima LM.Specificity in DNA recognition by a peptide from papillomavirus E2 protein.FEBS Letters,2006,580(8):1919-1924.
[11].Guilherme Menegon Giesel, Lui's Mauri'cio T.R. Lima, Joana Faber-Barata,Characterization of the papillomavirus a1E2 peptide unfolded to folded transition upon DNA binding, FEBS Letters, 2008,582:3619-3624.
[12]Oh KJ,Kalinina A,Bagchi S.Destabilization of Rb by human papillomavirus E7 is cell cycle dependent:E2-25K is involved in the proteolysis[J].Virology.2010,396(1):118-124.
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