Whole Genome Analysis of Human Papillomavirus Type 16 Multiple Infection in Cervical Cancer Patients

  • Chansaenroj, Jira (Center of Excellence in Clinical Virology, Department of Paediatrics, Faculty of Medicine, Chulalongkorn University) ;
  • Theamboonlers, Apiradee (Center of Excellence in Clinical Virology, Department of Paediatrics, Faculty of Medicine, Chulalongkorn University) ;
  • Junyangdikul, Pairoj (Department of Pathology, Samitivej Srinakharin Hospital) ;
  • Swangvaree, Sukumarn (Department of Gynecologic Oncology, National Cancer Institute) ;
  • Karalak, Anant (Department of Pathology, National Cancer Institute) ;
  • Poovorawan, Yong (Center of Excellence in Clinical Virology, Department of Paediatrics, Faculty of Medicine, Chulalongkorn University)
  • Published : 2012.02.29


The characterization of the whole genome of human papillomavirus type 16 (HPV16) from cervical cancer specimens with multiple infections in comparison with single infection samples as the oncogenic potential of the virus may differ. Cervical carcinoma specimens positive for HPV16 by PCR and INNO-LiPA were randomly selected for whole genome characterization. Two HPV16 single infection and six HPV16 multiple infection specimens were subjected to whole genome analysis by using conserved primers and subsequent sequencing. All HPV16 whole genomes from single infection samples clustered in the European (E) lineage while all multiple infection specimens belonged to the non-European lineage. The variations in nucleotide sequences in E6, E7, E2, L1 and Long control region (LCR) were evaluated. In the E6 region, amino acid changes at L83V were related to increased cancer progression. An amino acid variation N29S within the E7 oncoprotein significantly associated with severity of lesion was also discovered. In all three domains of the E2 gene non synonymous mutations were found. The L1 region showed various mutations which may be related to conformation changes of viral epitopes. Some transcription factor binding sites in the LCR region correlated to virulence were shown on GRE/1, TEF-1, YY14 and Oct-1. HPV16 European variant prone to single infection may harbor a major variation at L83V which significantly increases the risk for developing cervical carcinoma. HPV16 non-European variants prone to multiple infections may require many polymorphisms to enhance the risk of cervical cancer development.


  1. Andersson S, Alemi M, Rylander E, et al (2000). Uneven distribution of HPV 16 E6 prototype and variant (L83V) oncoprotein in cervical neoplastic lesions. Br J Cancer, 83, 307-10.
  2. Bernard HU, Chan SY, Manos MM, et al (1994). Identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms. J Infect Dis, 170, 1077-85.
  3. Bhattacharjee B, Sengupta S (2006). HPV16 E2 gene disruption and polymorphisms of E2 and LCR: some significant associations with cervical cancer in Indian women. Gynecol Oncol, 100, 372-8.
  4. Bosch FX, Manos MM, Munoz N, et al (1995). Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst, 87, 796-802.
  5. Broccolo F, Drago F, Careddu AM, et al (2005). Additional evidence that pityriasis rosea is associated with reactivation of human herpesvirus-6 and -7. J Invest Dermatol, 124, 1234-40.
  6. Burk RD, Chen Z, Van Doorslaer K (2009). Human papillomaviruses: genetic basis of carcinogenicity. Public Health Genomics, 12, 281-90.
  7. 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.<449::AID-IJC3>3.0.CO;2-0
  8. Chakrabarti O, Veeraraghavalu K, Tergaonkar V, et al (2004). Human papillomavirus type 16 E6 amino acid 83 variants enhance E6-mediated MAPK signaling and differentially regulate tumorigenesis by notch signaling and oncogenic Ras. J Virol, 78, 5934-45.
  9. Durst M, Glitz D, Schneider A, zur Hausen H (1992). Human papillomavirus type 16 (HPV 16) gene expression and DNA replication in cervical neoplasia: analysis by in situ hybridization. Virology, 189, 132-40.
  10. Frati E, Bianchi S, Colzani D, et al (2011). Genetic variability in the major capsid L1 protein of human papillomavirus type 16 (HPV-16) and 18 (HPV-18). Infect Genet Evol, 11, 2119-24.
  11. Hall TA (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser, 41,95-98.
  12. Hildesheim A, Schiffman M, Bromley C, et al (2001). Human papillomavirus type 16 variants and risk of cervical cancer. J Natl Cancer Inst, 93, 315-8.
  13. Hildesheim A, Wang SS (2002). Host and viral genetics and risk of cervical cancer: a review. Virus Res, 89, 229-40.
  14. Icenogle JP, Sathya P, Miller DL, Tucker RA, Rawls WE (1991). Nucleotide and amino acid sequence variation in the L1 and E7 open reading frames of human papillomavirus type 6 and type 16. Virology, 184, 101-7.
  15. Jeon S, Allen-Hoffmann BL, Lambert PF (1995). Integration of human papillomavirus type 16 into the human genome correlates with selective growth advantage of cells. J Virol, 69, 2989-97.
  16. Kammer C, Tommasino M, Syrjänen S, et al (2002). Variants of the long control region and the E6 oncogene in European human papillomavirus type 16 isolates: implications for cervical disease. Br J Cancer, 86, 269-73.
  17. Kurvinen K, Yliskoski M, Saarikoski S, Syrjänen K, Syrjänen S (2000). Variants of the long control region of human papillomavirus type 16. Eur J Cancer, 36, 1402-10.
  18. Li N, Franceschi S, Howell-Jones R, Snijders PJF, Clifford GM (2011). Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: variation by geographical region, histological type and year of publication. Int J Cancer, 128, 927-35.
  19. Lizano M, Berumen J, Garcia-Carranca A (2009). HPV-related carcinogenesis: basic concepts, viral types and variants. Arch Med Res, 40, 428-34.
  20. Londesborough P, Ho L, Terry G, et al (1996). Human papillomavirus genotype as a predictor of persistence and development of high-grade lesions in women with minor cervical abnormalities. Int J Cancer, 69, 364-8.<364::AID-IJC2>3.0.CO;2-3
  21. Lurchachaiwong W, Junyangdikul P, Payungporn S, et al (2009). Relationship between hybrid capture II ratios and DNA amplification of E1, E6 and L1 genes used for the detection of human papillomavirus in samples with different cytological findings. Asian Pac J Allergy Immunol, 27, 217-24.
  22. Lurchachaiwong W, Junyangdikul P, Payungporn S, et al (2009). Entire genome characterization of human papillomavirus type 16 from infected Thai women with different cytological findings. Virus Genes, 39, 30-8.
  23. Mantovani F., Banks L (2001). The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene, 20, 7874-87.
  24. Munoz N, Castellsague X, de Gonzalez AB, Gissmann L (2006). Chapter 1: HPV in the etiology of human cancer. Vaccine, 24, 1-10.
  25. Myers G, et al (1997). Human papillomaviruses 1997: a compilation and analysis of nucleic acid and amino acid sequences. Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, NM.
  26. Pastrana DV, Vass WC, Lowy DR, Schiller JT (2001). NHPV16 VLP vaccine induces human antibodies that neutralize divergent variants of HPV16. Virology, 279, 361-9.
  27. Phelps WC, Münger K, Yee CL, Barnes JA, Howley PM (1992). Structure-function analysis of the human papillomavirus type 16 E7 oncoprotein. J Virol, 66, 2418-27.
  28. Pillai MR, Hariharan R, Babu JM, et al (2009). Molecular variants of HPV-16 associated with cervical cancer in Indian population. Int J Cancer, 125, 91-103.
  29. Pista A, Oliveira A, Barateiro A, et al (2007). Molecular variants of human papillomavirus type 16 and 18 and risk for cervical neoplasia in Portugal. J Med Virol, 79, 1889-97.
  30. Shang Q, Wang Y, Fang Y, et al (2011). Human papillomavirus type 16 variant analysis of E6, E7, and L1 genes and long control region in cervical carcinomas in patients in northeast China. J Clin Microbiol, 49, 2656-63.
  31. Sibbet GJ, Cuthill S, Campo MS (1995). The enhancer in the long control region of human papillomavirus type 16 is up regulated by PEF-1 and down regulated by Oct-1. J Virol, 69, 4006-11.
  32. Sichero L, Ferreira S, Trottier H, et al (2007). High grade cervical lesions are caused preferentially by non-European variants of HPVs 16 and 18. Int J Cancer, 120, 1763-8.
  33. Smith B, Chen Z, Reimers L, et al (2011). Sequence Imputation of HPV16 Genomes for Genetic Association Studies. PLoS One, 6, 21375-?.
  34. Smith JS, Lindsay L, Hoots B, et al (2007). Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. Int J Cancer, 121, 621-32.
  35. Song YS, Kee SH, Kim JW, et al (1997). Major sequence variants in E7 gene of human papillomavirus type 16 from cervical cancerous and noncancerous lesions of Korean women. Gynecol Oncol, 66, 275-81.
  36. Spinillo A, Dal Bello B, Gardella B, et al (2009). Multiple human papillomavirus infection and high grade cervical intraepithelial neoplasia among women with cytological diagnosis of atypical squamous cells of undetermined significance or low grade squamous intraepithelial lesions. Gynecol Oncol, 113, 115-9.
  37. Stewart AC, Eriksson AM, Manos MM, et al (1996). Intratype variation in 12 human papillomavirus types: a worldwide perspective. J Virol, 70, 3127-36.
  38. Stöppler MC, Ching K, Stöppler H, et al (1996). Natural variants of the human papillomavirus type 16 E6 protein differ in their abilities to alter keratinocyte differentiation and to induce p53 degradation. J Virol, 70, 6987-93.
  39. Stunkel W, Bernard HU (1999). The chromatin structure of the long control region of human papillomavirus type 16 represses viral oncoprotein expression. J Virol, 73, 1918-30.
  40. Sun Z, Ren G, Cui X, et al (2011). Genetic diversity of HPV-16 E6, E7, and L1 genes in women with cervical lesions in Liaoning Province, China. Int J Gynecol Cancer, 21, 551-8.
  41. Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol, 24, 1596-99.
  42. Tan SH, Leong LEC, 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.
  43. Tan SH, Bartsch D, Schwarz E, Bernard HU (1998). Nuclear matrix attachment regions of human papillomavirus type 16 point toward conservation of these genomic elements in all genital papillomaviruses. J Virol, 72, 3610-22.
  44. Tornesello ML, Buonaguro FM, Buonaguro L, et al (2000). Identification and functional analysis of sequence rearrangements in the long control region of human papillomavirus type 16 Af-1 variants isolated from Ugandan penile carcinomas. J Gen Virol, 81, 2969-82
  45. 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.
  46. Veress G, Murvai M, Szarka K, et al (2001). Transcriptional activity of human papillomavirus type 16 variants having deletions in the long control region. Eur J Cancer, 37, 1946-52.
  47. Villa LL, Sichero L, Rahal P, et al (2000). Molecular variants of human papillomavirus types 16 and 18 preferentially associated with cervical neoplasia. J Gen Virol, 81, 2959-68.
  48. Watts KJ, Thompson CH, Cossart YE, Rose BR (2001). Variable oncogene promoter activity of human papillomavirus type 16 cervical cancer isolates from Australia. J Clin Microbiol, 39, 2009-14.
  49. Xi LF, Koutsky LA, Galloway DA, et al (1997). Genomic variation of human papillomavirus type 16 and risk for high grade cervical intraepithelial neoplasia. J Natl Cancer Inst, 89, 796-802.
  50. Yamada T, Wheeler CM, Halpern AL, et al (1995). Human papillomavirus type 16 variant lineages in United States populations characterized by nucleotide sequence analysis of the E6, L2, and L1 coding segments. J Virol, 69, 7743-53.
  51. 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.
  52. Zuna RE, Moore WE, Shanesmith RP et al (2009). Association of HPV16 E6 variants with diagnostic severity in cervical cytology samples of 354 women in a US population. Int J Cancer, 125, 2609-13.

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