Skip to main content
SpringerLink
Log in

Molecular and phylogenetic analysis of the HPV 16 E4 gene in cervical lesions from women in Greece

  • Original Article
  • Published:
Archives of Virology Aims and scope Submit manuscript

Abstract

The HPV16 E1∧E4 protein is thought to contribute to the release of newly formed viral particles from infected epithelia. In order to investigate amino acid mutations in the HPV16 E1∧E4 protein, the complete E4 ORF was amplified by PCR in 27 HPV16-positive cervical samples, and the amplicons were cloned. Fifteen nucleic acid variations were identified in the E4 ORF, including seven silent nucleic acid mutations. In addition, nine amino acid mutations (A7V, A7P, L16I, D45E, L59I, L59T, Q66P, S72F, H75Q) were detected in the E1∧E4 protein, and these were associated with the severity of cervical malignancy. A maximum-likelihood phylogenetic tree was constructed based on the E4 ORF, and nucleotide sequence analysis of the E4, E6 and E7 genes from the same samples was conducted in order to determine the phylogenetic origin of the cloned sequences from the amplified HPV16 E4. Based on the nucleotide sequence and phylogenetic analysis it was revealed that even though E4 ORF constitutes a small polymorphic portion of the viral genome (288 bp), it could provide valuable information about the origins of the HPV16 genome. In addition, molecular evolutionary analysis of the E4 coding region revealed that neutral selection is dominant in the overlapping region of the E4 and E2 ORFs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  1. Bernard HU, Burk RD, Chen Z, van Doorslaer K, Hausen H, de Villiers EM (2010) Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 401:70–79

    Article  PubMed  CAS  Google Scholar 

  2. Sundström K, Eloranta S, Sparén P, Arnheim Dahlström L, Gunnell A, Lindgren A, Palmgren J et al (2010) Prospective study of human papillomavirus (HPV) types, HPV persistence, and risk of squamous cell carcinoma of the cervix. Cancer Epidemiol Biomarkers Prev 19:2469–2478

    Article  PubMed  Google Scholar 

  3. Castle PE, Schiffman M, Wheeler CM, Wentzensen N, Gravitt PE (2010) Human papillomavirus genotypes in cervical intraepithelial neoplasia grade 3. Cancer Epidemiol Biomarkers Prev 19:1675–1681

    Article  PubMed  Google Scholar 

  4. Bruni L, Diaz M, Castellsagué X, Ferrer E, Bosch FX, de Sanjosé S (2010) Cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. J Infect Dis 202:1789–1799

    Article  PubMed  Google Scholar 

  5. Kedzia W, Józefiak A, Pruski D, Rokita W, Marek S (2010) Human papilloma virus genotyping in women with CIN 1. Ginekol Pol 81:664–667

    PubMed  Google Scholar 

  6. Tornesello ML, Losito S, Benincasa G, Fulciniti F, Botti G, Greggi S, Buonaguro L, Buonaguro FM (2011) Human papillomavirus (HPV) genotypes and HPV16 variants and risk of adenocarcinoma and squamous cell carcinoma of the cervix. Gynecol Oncol 121:32–42

    Article  PubMed  CAS  Google Scholar 

  7. Yamada T, Manos MM, Peto J, Greer CE, Munoz N, Bosch FX, Wheeler CM (1997) Human papillomavirus type 16 sequence variation in cervical cancers: a worldwide perspective. J Virol 71:2463–2472

    PubMed  CAS  Google Scholar 

  8. Yamada T, Wheeler CM, Halpern AL, Stewart AC, Hildesheim A, Jenison SA (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 69:7743–7753

    PubMed  CAS  Google Scholar 

  9. Wheeler CM, Yamada T, Hildesheim A, Jenison SA (1997) Human papillomavirus type 16 sequence variants: identification by E6 and L1 lineage-specific hybridization. J Clin Microbiol 35:11–19

    PubMed  CAS  Google Scholar 

  10. Tsakogiannis D, Ruether IG, Kyriakopoulou Z, Pliaka V, Theoharopoulou A, Skordas V, Panotopoulou E, Nepka C, Markoulatos P (2012) Sequence variation analysis of the E2 gene of human papilloma virus type 16 in cervical lesions from women in Greece. Arch Virol. doi:10.1007/s00705-012-1236-8

    Google Scholar 

  11. Swan DC, Rajeevan M, Tortolero-Luna G, Follen M, Tucker RA, Unger ER (2005) Human papillomavirus type 16 E2 and E6/E7 variants. Gynecol Oncol 96:695–700

    Article  PubMed  CAS  Google Scholar 

  12. Eriksson A, Herron JR, Yamada T, Wheeler CM (1999) Human papillomavirus type 16 variant lineages characterised by nucleotide sequence analysis of the E5 coding segment and the E2 hinge region. J Gen Virol 80:595–600

    PubMed  CAS  Google Scholar 

  13. Sichero L, Ferreira S, Trottier H, Duarte-Franco E, Ferenczy A, Franco EL, Villa LL (2007) High grade cervical lesions are caused preferentially by non-European variants of HPVs 16 and 18. Int J Cancer 120:1763–1768

    Article  PubMed  CAS  Google Scholar 

  14. Sanchez GI, Kleter B, Gheit T, van Doorn LJ, de Koning MN, de Sanjosé S et al (2011) Clinical evaluation of polymerase chain reaction reverse hybridization assay for detection and identification of human papillomavirus type 16 variants. J Clin Virol 51:165–169

    Article  PubMed  CAS  Google Scholar 

  15. Tornesello ML, Losito S, Benincasa G, Fulciniti F, Botti G, Greggi S et al (2011) Human papillomavirus (HPV) genotypes and HPV16 variants and risk of adenocarcinoma and squamous cell carcinoma of the cervix. Gynecol Oncol 121:32–42

    Article  PubMed  CAS  Google Scholar 

  16. Lehoux M, D’Abramo CM, Archambault J (2009) Molecular mechanisms of human papillomavirus-induced carcinogenesis. Public Health Genomics. 12:268–280

    Article  PubMed  Google Scholar 

  17. Doorbar J (2007) Papillomavirus life cycle organization and biomarker selection. Dis Markers 23:297–313

    PubMed  CAS  Google Scholar 

  18. Snijders PJ, Steenbergen RD, Heideman DA, Meijer CJ (2006) HPV-mediated cervical carcinogenesis: concepts and clinical implications. J Pathol 208:152–164

    Article  PubMed  CAS  Google Scholar 

  19. Münger K, Baldwin A, Edwards KM, Hayakawa H, Nguyen CL, Owens M, Grace M, Huh K (2004) Mechanisms of human papillomavirus-induced oncogenesis. J Virol 78:11451–11460

    Article  PubMed  Google Scholar 

  20. Fehrmann F, Laimins LA (2003) Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation. Oncogene 22:5201–5207

    Article  PubMed  CAS  Google Scholar 

  21. Schwartz S (2008) HPV-16 RNA processing. Front Biosci 13:5880–5891

    Article  PubMed  CAS  Google Scholar 

  22. Schwartz S, Zhao S, Öberg D, Rush M (2004) Regulation of papillomavirus late gene expression. Recent Res Dev Virol 6:29–45

    CAS  Google Scholar 

  23. Doorbar J, Foo C, Coleman N, Medcalf L, Hartley O, Prospero T et al (1997) Characterization of events during the late stages of HPV16 infection in vivo using high-affinity synthetic Fabs to E4. Virology 238:40–52

    Article  PubMed  CAS  Google Scholar 

  24. Middleton K, Peh W, Southern S, Griffin H, Sotlar K, Nakahara T et al (2003) Organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers. J Virol 77:10186–10201

    Article  PubMed  CAS  Google Scholar 

  25. Chow LT, Broker TR (2007) Human papillomavirus RNA transcription. In: Garcea RL, DiMaio D (eds) The Papillomaviruses. Springer, New York, pp 109–144

    Google Scholar 

  26. Howley PM (1996) Papillomavirinae: the viruses and their replication. In: Fields BN, Knipe DM, Howley PM (eds) Fields virology, 3rd edn. Lippincott-Raven, Philadelphia, pp 2045–2076

    Google Scholar 

  27. McIntosh PB, Laskey P, Sullivan K, Davy C, Wang Q, Jackson DJ, Griffin HM, Doorbar J (2010) E1–E4-mediated keratin phosphorylation and ubiquitylation: a mechanism for keratin depletion in HPV16-infected epithelium. J Cell Sci 123:2810–2822

    Article  PubMed  CAS  Google Scholar 

  28. Davy CE, Jackson DJ, Wang Q, Raj K, Masterson PJ, Fenner NF et al (2002) Identification of a G(2) arrest domain in the E1 wedge E4 protein of human papillomavirus type 16. J Virol 76:9806–9818

    Article  PubMed  CAS  Google Scholar 

  29. Davy CE, Jackson DJ, Raj K, Peh WL, Southern SA, Das P, Sorathia R et al (2005) Human papillomavirus type 16 E1 E4-induced G2 arrest is associated with cytoplasmic retention of active Cdk1/cyclin B1 complexes. J Virol 79:3998–4011

    Article  PubMed  CAS  Google Scholar 

  30. Roberts S, Kingsbury SR, Stoeber K, Knight GL, Gallimore PH, Williams GH (2008) Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells. J Virol 82:9056–9064

    Article  PubMed  CAS  Google Scholar 

  31. Davy C, McIntosh P, Jackson DJ, Sorathia R, Miell M, Wang Q, Khan J, Soneji Y, Doorbar J (2009) A novel interaction between the human papillomavirus type 16 E2 and E1–E4 proteins leads to stabilization of E2. Virology 394:266–275

    Article  PubMed  CAS  Google Scholar 

  32. Casas I, Powell L, Klapper PE, Cleator GM (1995) New method for the extraction of viral RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J Virol Methods 53:25–36

    Article  PubMed  CAS  Google Scholar 

  33. Puranen M, Saarikoski S, Syrjänen K, Syrjänen S (1996) Polymerase chain reaction amplification of human papillomavirus DNA from archival, Papanicolaou-stained cervical smears. Acta Cytol 40:391–395

    Article  PubMed  CAS  Google Scholar 

  34. Sotlar K, Diemer D, Dethleffs A, Hack Y et al (2004) Detection and typing of human papillomavirus by E6 nested multiplex PCR. J Clin Microbiol 42:3176–3184

    Article  PubMed  CAS  Google Scholar 

  35. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  PubMed  CAS  Google Scholar 

  36. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Article  PubMed  CAS  Google Scholar 

  37. Pond SL, Frost SD (2005) Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 21:2531–2533

    Article  PubMed  CAS  Google Scholar 

  38. Suzuki Y, Gojobori T (1999) A method for detecting positive selection at single amino acid sites. Mol Biol Evol 16:1315–1328

    Article  PubMed  CAS  Google Scholar 

  39. Drummond AJ, Rambaut A (2007) BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7:214

    Article  PubMed  Google Scholar 

  40. Doorbar, J and Myers G (1996) The E4 protein. In: Myers HDG, Icenogelet J (eds) Human papillomaviruses, vol. 3. Los Alamos National Laboratory, Los Alamos, NM, pp 58–80

  41. Eriksson A, Herron JR, Yamada T, Wheeler CM (1999) Human papillomavirus type 16 variant lineages characterized by nucleotide sequence analysis of the E5 coding segment and the E2 hinge region. J Gen Virol 80:595–600

    PubMed  CAS  Google Scholar 

  42. Roberts S, Ashmole I, Gibson LJ, Rookes SM, Barton GJ, Gallimore PH (1994) Mutational analysis of human papillomavirus E4 proteins: identification of structural features important in the formation of cytoplasmic E4/cytokeratin networks in epithelial cells. J Virol 68:6432–6445

    PubMed  CAS  Google Scholar 

  43. Wang Q, Griffin H, Southern S, Jackson D, Martin A et al (2004) Functional analysis of the human papillomavirus type 16 E1=E4 protein provides a mechanism for in vivo and in vitro keratin filament reorganization. J Virol 78:821–833

    Article  PubMed  CAS  Google Scholar 

  44. Hughes AL, Hughes MA (2005) Patterns of nucleotide difference in overlapping and non-overlapping reading frames of papillomavirus genomes. Virus Res 113:81–88

    Article  PubMed  CAS  Google Scholar 

  45. Burk RD, Chen Z, Van Doorslaer K (2009) Human papillomaviruses: genetic basis of carcinogenicity. Public Health Genomics 12:281–290

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by research grants of the Postgraduate Programmes ‘Applications of Molecular Biology-Genetics. Diagnostic Biomarkers’, code 3817, and “Biotechnology”, code 3439, of the University of Thessaly, School of Health Sciences, Department of Biochemistry and Biotechnology.

Conflict of interest

All authors declare that they have no conflicting or dual interests.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biotechnology, Microbiology-Virology Laboratory, School of Health Sciences, University of Thessaly, Ploutonos 26 and Aiolou, 41221, Larissa, Greece

    D. Tsakogiannis, I. G. A. Ruether, Z. Kyriakopoulou, V. Pliaka, V. Skordas & P. Markoulatos

  2. Department of Microbiology, Medical School, University of Ioannina, Ioannina, Greece

    C. Gartzonika & S. Levidiotou-Stefanou

Authors
  1. D. Tsakogiannis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. G. A. Ruether
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Z. Kyriakopoulou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. V. Pliaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. V. Skordas
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C. Gartzonika
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S. Levidiotou-Stefanou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. P. Markoulatos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P. Markoulatos.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tsakogiannis, D., Ruether, I.G.A., Kyriakopoulou, Z. et al. Molecular and phylogenetic analysis of the HPV 16 E4 gene in cervical lesions from women in Greece. Arch Virol 157, 1729–1739 (2012). https://doi.org/10.1007/s00705-012-1356-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00705-012-1356-1

Keywords

Search

Navigation