DOI QR코드

DOI QR Code

Polymorphisms and Functional Analysis of the Intact Human Papillomavirus16 E2 Gene

  • Ekalaksananan, Tipaya (Department of Microbiology, Faculty of Medicine, Khon Kaen University) ;
  • Jungpol, Watcharapol (Department of Microbiology, Faculty of Medicine, Khon Kaen University) ;
  • Prasitthimay, Chuthamas (Department of Microbiology, Faculty of Medicine, Khon Kaen University) ;
  • Wongjampa, Weerayut (Department of Microbiology, Faculty of Medicine, Khon Kaen University) ;
  • Kongyingyoes, Bunkerd (Department of Pharmacology, Faculty of Medicine, Khon Kaen University) ;
  • Pientong, Chamsai (Department of Microbiology, Faculty of Medicine, Khon Kaen University)
  • Published : 2015.01.06

Abstract

High risk human papillomavirus (HR-HPV) E2 proteins play roles in transcriptional regulation and are commonly functionally disrupted when the HPV genome integrates into host chromosomes. Some 15-40% of cancer cases, however, contain an intact E2 gene or episomal HPV. In these cases, polymorphism of the E2 gene might be involved. This study aimed to determine polymorphisms of the E2 gene in episomal HPV16 detected in high grade squamous intraepithelial lesions and squamous cell carcinomas and altered functions compared to the E2 prototype. The E2 gene was amplified and sequenced. Two expression vectors containing E2 gene polymorphisms were constructed and transfected in SiHa and C33A cells, then E6 gene as well as Il-10 and TNF-${\alpha}$ expression was determined by quantitative RT-PCR. Expression vectors and reporter vectors containing the HPV16 long control region (LCR) were co-transfected and transcriptional activity was determined. The results showed that a total of 32 nucleotides and 23 amino acids were changed in all 20 cases of study, found in the transactivation (TA) domain, hinge (H) region and DNA binding (DB) domain with 14, 5 and 13 nucleotide positions. They mostly caused amino acid change. The expressing vectors containing different E2 gene polymorphisms showed E6 mRNA suppression, TNF-${\alpha}$ mRNA suppression and IL-10 induction but no statistically significant differences when compared to the E2 prototype. Moreover, promoter activity in HPV16 LCR was not affected by E2 protein with different gene polymorphisms, in contrast to nucleotide variations in LCR that showed an effect on transcription activity. These results demonstrated that E2 gene polymorphisms of episomal HPV16 did not affect transcriptional regulation and suggested that nucleotide variation as well as epigenetic modification of the LCR might play a role in inducing malignant transformation of cells containing episomal HPV16.

Keywords

Cervical cancer;human papillomavirus;E2 protein;polymorphism;function

Acknowledgement

Supported by : Khon Kaen University

References

  1. Bermudez-Morales VH, Gutierrez LX, Alcocer-Gonzalez JM, Burguete A, Madrid-Marina V (2008). Correlation between IL-10 gene expression and HPV infection in cervical cancer: a mechanism for immune response escape. Cancer Invest, 26, 1037-43. https://doi.org/10.1080/07357900802112693
  2. Bermudez-Morales VH, Peralta-Zaragoza O, Alcocer-Gonzalez JM, Moreno J, Madrid-Marina V (2011). IL-10 expression is regulated by HPV E2 protein in cervical cancer cells. Mol Med Rep, 4, 369-75.
  3. Bhattacharjee B, Sengupta S (2006). CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2. Virology, 354, 280-5. https://doi.org/10.1016/j.virol.2006.06.018
  4. Blakaj DM, Fernandez-Fuentes N, Chen Z, et al (2009). Evolutionary and biophysical relationships among the papillomavirus E2 proteins. Front Biosci, 14, 900-17.
  5. Casas L, Galvan SC, Ordonez RM, et al (1999). Asian-American variants of human papillomavirus type 16 have extensive mutations in the E2 gene and are highly amplified in cervical carcinomas. Int J Cancer, 83, 449-55. https://doi.org/10.1002/(SICI)1097-0215(19991112)83:4<449::AID-IJC3>3.0.CO;2-0
  6. Chaiwongkot A, Vinokurova S, Pientong C, et al (2013). Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions. Int J Cancer, 132, 2087-94. https://doi.org/10.1002/ijc.27906
  7. Chen Z, Storthz KA, Shillitoe EJ (1997). Mutations in the long control region of human papillomavirus DNA in oral cancer cells, and their functional consequences. Cancer Res, 57, 1614-9.
  8. Chopjitt P, Ekalaksananan T, Pientong C, et al (2009). Prevalence of human papillomavirus type 16 and its variants in abnormal squamous cervical cells in Northeast Thailand. Int J Infect Dis, 13, 212-9. https://doi.org/10.1016/j.ijid.2008.06.017
  9. Cornet I, Gheit T, Franceschi S, et al (2012). Human papillomavirus type 16 genetic variants: phylogeny and classification based on E6 and LCR. J Virol, 86, 6855-61. https://doi.org/10.1128/JVI.00483-12
  10. de Sanjose S, Quint WG, Alemany L, et al (2010). Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol, 11, 1048-56. https://doi.org/10.1016/S1470-2045(10)70230-8
  11. Demeret C, Yaniv M, Thierry F (1994). The E2 transcriptional repressor can compensate for Sp1 activation of the human papillomavirus type 18 early promoter. J Virol, 68, 7075-82.
  12. Doeberitz Mv, Vinokurova S (2009). Host factors in HPV-related carcinogenesis: cellular mechanisms controlling HPV infections. Arch Med Res, 40, 435-42. https://doi.org/10.1016/j.arcmed.2009.06.002
  13. Gammoh N, Gardiol D, Massimi P, Banks L (2009). The Mdm2 ubiquitin ligase enhances transcriptional activity of human papillomavirus E2. J Virol, 83, 1538-43. https://doi.org/10.1128/JVI.01551-08
  14. Grm HS, Massimi P, Gammoh N, Banks L (2005). Crosstalk between the human papillomavirus E2 transcriptional activator and the E6 oncoprotein. Oncogene, 24, 5149-64. https://doi.org/10.1038/sj.onc.1208701
  15. Helfer CM, Wang R, You J (2013). Analysis of the papillomavirus E2 and bromodomain protein Brd4 Interaction using bimolecular fluorescence complementation. PloS One, 8, 77994. https://doi.org/10.1371/journal.pone.0077994
  16. Jacquin E, Baraquin A, Ramanah R, et al (2013). Methylation of human papillomavirus type 16 CpG sites at E2-binding site 1 (E2BS1), E2BS2, and the Sp1-binding site in cervical cancer samples as determined by high-resolution melting analysis-PCR. J Clin Microbiol, 51, 3207-15. https://doi.org/10.1128/JCM.01106-13
  17. Kadaja M, Silla T, Ustav E, Ustav M (2009). Papillomavirus DNA replication-from initiation to genomic instability. Virology, 384, 360-8. https://doi.org/10.1016/j.virol.2008.11.032
  18. Lee D, Kim JW, Kim K, et al (2002). Functional interaction between human papillomavirus type 18 E2 and poly (ADPribose) polymerase 1. Oncogene, 21, 5877-85. https://doi.org/10.1038/sj.onc.1205723
  19. Moody CA, Laimins LA (2010). Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Cancer, 10, 550-60. https://doi.org/10.1038/nrc2886
  20. Nagao S, Yoshinouchi M, Miyagi Y, et al (2002). Rapid and sensitive detection of physical status of human papillomavirus type 16 DNA by quantitative real-time PCR. J Clin Microbiol, 40, 863-7. https://doi.org/10.1128/JCM.40.3.863-867.2002
  21. Natphopsuk S, Settheetham-Ishida W, Pientong C, et al (2013). Human papillomavirus genotypes and cervical cancer in northeast Thailand. Asian Pac J Cancer Prev, 14, 6961-4. https://doi.org/10.7314/APJCP.2013.14.11.6961
  22. Nishimura A, Ono T, Ishimoto A, et al (2000). Mechanisms of human papillomavirus E2-mediated repression of viral oncogene expression and cervical cancer cell growth inhibition. J Virol, 74, 3752-60. https://doi.org/10.1128/JVI.74.8.3752-3760.2000
  23. Oliveira JG, Colf LA, McBride AA (2006). Variations in the association of papillomavirus E2 proteins with mitotic chromosomes. Proc Natl Acad Sci USA, 103, 1047-52. https://doi.org/10.1073/pnas.0507624103
  24. Pett M, Coleman N (2007). Integration of high-risk human papillomavirus: a key event in cervical carcinogenesis? J Pathol, 212, 356-67. https://doi.org/10.1002/path.2192
  25. Pientong C, Wongwarissara P, Ekalaksananan T, et al (2013). Association of human papillomavirus type 16 long control region mutation and cervical cancer. Virol J, 10, 30-7. https://doi.org/10.1186/1743-422X-10-30
  26. Romanczuk H, Thierry F, Howley PM (1990). Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters. J Virol, 64, 2849-59.
  27. Shen XH, Liu SH (2013). Human papillomavirus genotypes associated with mucopurulent cervicitis and cervical cancer in Hangzhou, China. Asian Pac J Cancer Prev, 14, 3603-6. https://doi.org/10.7314/APJCP.2013.14.6.3603
  28. Steger G, Ham J, Lefebvre O, Yaniv M (1995). The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding. EMBO J, 14, 329-40.
  29. Tan SH, Leong LE, Walker PA, Bernard HU (1994). The human papillomavirus type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID. J Virol, 68, 6411-20.
  30. Veress G, Szarka K, Dong XP, Gergely L, Pfister H (1999). Functional significance of sequence variation in the E2 gene and the long control region of human papillomavirus type 16. J Gen Virol, 80, 1035-43. https://doi.org/10.1099/0022-1317-80-4-1035
  31. Walboomers JM, Jacobs MV, Manos MM, et al (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol, 189, 12-9. https://doi.org/10.1002/(SICI)1096-9896(199909)189:1<12::AID-PATH431>3.0.CO;2-F
  32. Wang L, Dai SZ, Chu HJ, Cui HF, Xu XY (2013). Integration sites and genotype distributions of human papillomavirus in cervical intraepithelial neoplasia. Asian Pac J Cancer Prev, 14, 3837-41. https://doi.org/10.7314/APJCP.2013.14.6.3837
  33. Yamada T, Manos MM, Peto J, et al (1997). Human papillomavirus type 16 sequence variation in cervical cancers: a worldwide perspective. J Virol, 71, 2463-72.
  34. Yao JM, Breiding DE, Androphy EJ (1998). Functional interaction of the bovine papillomavirus E2 transactivation domain with TFIIB. J Virol, 72, 1013-9.
  35. Zou N, Lin BY, Duan F, et al (2000). The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association. J Virol, 74, 3761-70. https://doi.org/10.1128/JVI.74.8.3761-3770.2000

Cited by

  1. Integration of human papillomavirus 16 in esophageal carcinoma samples vol.12, pp.1, 2017, https://doi.org/10.1186/s13027-017-0164-3
  2. Intratype variants of the E2 protein from human papillomavirus type 18 induce different gene expression profiles associated with apoptosis and cell proliferation pp.1432-8798, 2019, https://doi.org/10.1007/s00705-018-04124-6