Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

HPV-16 E1, E2 and E6 each complement the Ad5 helper gene set, increasing rAAV2 and wt AAV2 production

Gene Therapy volume 19, pages 418–424 (2012)Cite this article

Abstract

Adeno-associated virus type 2 (AAV) is a popular vector for human gene therapy, because of its safety record and ability to express genes long term. Yet large-scale recombinant (r) AAV production remains problematic because of low particle yield. The adenovirus (Ad) and herpes (simplex) virus helper genes for AAV have been widely used and studied, but the helper genes of human papillomavirus (HPV) have not. HPV-16 E1, E2 and E6 help wild-type (wt) AAV productive infection in differentiating keratinocytes, however, HEK293 cells are the standard cell line used for generating rAAV. Here we demonstrate that the three HPV genes were unable to stimulate significant rAAV replication in HEK293 cells when used alone. However, when used in conjunction (complementation) with the standard Ad5 helper gene set, E1, E2 and E6 were each capable of significantly boosting rAAV DNA replication and virus particle yield. Moreover, wt AAV DNA replication and virion yield were also significantly boosted by each HPV gene along with wt Ad5 virus co-infection. Mild-to-moderate changes in rep- and cap-encoded protein levels were evident in the presence of the E1, E2 and E6 genes. Higher wt AAV DNA replication was not matched by similar increases in the levels of rep-encoded protein. Moreover, although rep mRNA was upregulated, cap mRNA was upregulated more. Higher virus yields did correlate most consistently with increased Rep52-, VP3- and VP-related 21/31 kDa species. The observed boost in wt and rAAV production by HPV genes was not unexpected, as the Ad and HPV helper gene sets do not seem to recapitulate each other. These results raise the possibility of generating improved helper gene sets derived from both the Ad and HPV helper gene sets.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Hermonat PL, Muzyczka N . Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA 1984; 81: 6466–6470.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Jiang H, Pierce GF, Ozelo MC, de Paula EV, Vargas JA, Smith P et al. Evidence of multiyear factor IX expression by AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B. Mol Ther 2006; 14: 452–455.

    Article  CAS  PubMed  Google Scholar 

  3. You CX, Liu Y, Shi M, Cao M, Luo R-C, Hermonat PL . Comparison of AAV/IL-7 autocrine (T cell) versus paracrine (DC) gene delivery for enhancing CTL stimulation and function. Can Imm Immunother 2010; 59: 779–787.

    Article  CAS  Google Scholar 

  4. Khan JA, Cao M, Kang BY, Liu Y, Mehta JL, Hermonat PL . Systemic hNetrin-1 gene delivery by AAV lowers monocyte/macrophage accumulation and atherogenesis in vivo. Gene Therapy 2011; 18: 437–444.

    Article  CAS  PubMed  Google Scholar 

  5. Srivastava A, Lusby EW, Berns KI . Nucleotide sequence and organization of the adeno-associated 2 genome. J Virol 1983; 45: 555–564.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Hermonat PL, Labow MA, Wright R, Berns KI, Muzyczka N . Genetics of adeno-associated virus: isolation and preliminary characteriozation of adeno-associated virus type 2 mutants. J Virol 1984; 51: 319–339.

    Google Scholar 

  7. Tratschin JD, Miller IL, Carter BJ . Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol 198; 51: 611–619.

    Google Scholar 

  8. Samulski RJ, Srivastava A, Berns KI, Muzyczka N . Rescue of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci USA 1982; 79: 2077–2081.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Meyers C, Mane M, Kokorina N, Alam S, Hermonat PL . Ubiquitous human adeno-associated virus type 2 autonomously replicates in differentiating keratinocytes of a normal skin model. Virology 2000; 272: 338–346.

    Article  CAS  PubMed  Google Scholar 

  10. Meyers C, Alam S, Mane M, Hermonat PL . Altered biology ofadeno-associated virus type 2 and human papillomavirus during dual infection of natural host tissue. Virology 2001; 287: 30–39.

    Article  CAS  PubMed  Google Scholar 

  11. You H, Liu Y, Prasad CP, Agrawal N, Zhang D, Bandyopadhyay S et al. Multiple human papillomavirus genes affect the adeno-associated virus life cycle. Virology 2006; 344: 532–540.

    Article  CAS  PubMed  Google Scholar 

  12. Casto BC, Atchinson RW, Hammon WMcD . Studies on the relationship between adenoassociated virus type 1 and adenovirues. I. Replication of AAV-1 in certain cell cultures and its effect on helper adenovirus. Virology 1967; 18: 52–60.

    Article  Google Scholar 

  13. Buller RM, Janik JE, Subring ED, Rose JA . Herpes simplex virus type 1 and 2 completely help adeno-associated virus replication. J Virol 1981; 40: 241–247.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Chang L-S, Shenk T . Adeno-associated virus p5 promoter contains an adenovirus E1A inducible element and a binding site for the major late transcription factor. J Virol 1989; 63: 3479–3488.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Tratschin JD, West MHP, Sandbank T, Carter BJ . A human parvovirus, adenoassociated virus as a eukaryotic vector: transient expression and encapsidation of the prokaryotic gene for chloramphenicil acetyltransferase. Mol Cell Biol 1984; 4: 2072–2081.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Laughlin CA, Jones N, Carter BJ . Effects of deletions in adenovirus region 1 genes upon the replication of adeno-associated virus. J Virol 1982; 41: 868–876.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Chang L-S, Shenk T . The adenovirus DNA binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters. J Virol 1990; 64: 2103–2109.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Carter BJ, Marcus-Sekura CJ, Laughlin CA, Ketner G . Properties of an adenovirus type 2 mutant, Ad2dl807, having a deletion near the right-hand genome terminus: failure to help AAV replication. Virology 1983; 126: 505–516.

    Article  CAS  PubMed  Google Scholar 

  19. Richardson WD, Westphal WD . A cascade of adenovirus early functions are required for expression of adeno-associated virus. Cell 1981; 27: 131–141.

    Article  Google Scholar 

  20. Samulski RJ, Shenk T . Adenovirus E1B 55-M, polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNAs. J Virol 1988; 62: 206–210.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Huang M-M, Hearing P . Adenovirus early region 4 encodes two gene products with redundant effects in lytic infection. J Virol 1989; 63: 2605–2615.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Hart LS, Yannone SM, Naczki C, Orlando JS, Waters SB, Akman SA et al. The adenovirus E4orf6 protein inhibits DNA double strand break repair and radiosensitizes human tumor cells in an E1B-55K-independent manner. J Biol Chem 2005; 280: 1474–1481.

    Article  CAS  PubMed  Google Scholar 

  23. West MPH, Trempe JP, Tratschin JD, Carter BJ . Gene expression virus vectors: the effects of chimeric mRNA structure, helper virus, and adenovirus VA1 RNA. Virology 1987; 160: 38–47.

    Article  CAS  PubMed  Google Scholar 

  24. Janik JE, Huston MM, Cho K, Rose JA . Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA1 RNA. Virology 1989; 168: 320–329.

    Article  CAS  PubMed  Google Scholar 

  25. McPherson RA, Ginsburg HS, Rose JA . Adeno-associated virus helper activity of adenovirus DNA binding protein. J Virol 1982; 46: 523–529.

    Google Scholar 

  26. Strauss SE, Ginsburg HS, Rose JA . DNA-minus temperature sensitive mutants of adenovirus type 5 help-adeno-associated virus replication. J Virol 1976; 17: 140–148.

    Google Scholar 

  27. Jay FT, Laughlin CA, Carter BJ . Eukaryotic translational control: adeno-associated virus protein synthesis is affected by a mutation in the adenovirus DNA-binding protein. Proc Natl Acad Sci USA 1989; 78: 2827–2931.

    Google Scholar 

  28. Meyers MW, Carter BJ . Assembly of adeno-associated virus. Virology 1980; 102: 71–82.

    Article  Google Scholar 

  29. Richardson WD, Westphal WD . Requirement for either early region la or early region lb adenovirus gene products in the helper effect of adeno-associated virus J. Virol 1984; 10: 1–8.

    Google Scholar 

  30. Duan D, Sharma P, Dudus L, Zhang Y, Sanlioglu S, Yan Z et al. Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E40RF6 and E2a gene expression. J Virol 1999; 73: 161–1699.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Androphy EJ, Hubbert NL, Schiller JT, Lowy DR . Identification of the I-IPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines. EMBO J 1987; 6: 989–992.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Bedell MA, Jones KH, Grossman SG, Laimins LA . Identification of human papillomavirus type 18 transformation genes in immortalized and primary cells. J Virol 1989; 63: 1247–1255.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Del Vecchio AM, Romanczuk H, Howley PM, Baker CC . Transient replication of human papillomavirus DNAs. J Virol 1992; 66: 5949–5958.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Bandyopadhyay S, Raney KD, Liu Y, Hermonat PL . AAV-2 Rep78 and HPV-16 E1 interact in vitro, modulating their ATPase activity. Biochemistry 2008; 47: 845–856.

    Article  CAS  PubMed  Google Scholar 

  35. Bandyopadhyay S, Cao M, Liu Y, Hermonat PL . HPV E1 up-regulates replication-related biochemistries of AAV Rep78. Virology 2010; 402: 94–101.

    Article  CAS  PubMed  Google Scholar 

  36. Ustav M, Stenlund A . Transient replication of BPV- 1 requires two viral polypeptides encoded by the E1 and E2 open reading frames. EMBO J 1991; 10: 449–457.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  37. Sandier AB, Vande Pol SB, Spalholz BA . Repression of bovine papillomavirus type 1 transcription by the E1 replication protein. J Virol 1993; 67: 5079–5087.

    Google Scholar 

  38. Scheffner M, Romanczuk H, Monger K, Huibregtse JM, Mietz JA, Howley PM . Functions of human papillomavirus proteins. In: zur Hausen H (ed). Human Pathogenic Papillomaviruses. Springer-Verlag: Heidelberg, 1994, pp. 83–100.

    Chapter  Google Scholar 

  39. King JA, Dubielzig R, Grimm D, Kleinschmidt JA . DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids. EMBO J 2001; 20: 3282–3291.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Bonne-Andrea C, Santucci S, Clertant P, Tillier F . Bovine papillomavirus E1 protein binds specifically DNA polymerase alpha but not replication protein A. J Virol 1995; 69: 2341–2350.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Moody CA, Laimins LA . Human papillomavirus oncoproteins: pathways to transformation. Nat Rev Can 2010; 10: 550–560.

    Article  CAS  Google Scholar 

  42. Lambert PF . Papillomavirus DNA replication. J Virol 1991; 65: 3417–3420.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Horer M, Weger S, Butz K, Hoppe-Seyler F, Geisen C, Kleinschmidt JA . Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of homologous promoters. J Virol 1995; 69: 5485–5496.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Collaco RF, Cao X, Trempe JP . A helper virus-free packaging system for recombinant adeno-associated virus. Gene 1999; 238: 397–405.

    Article  CAS  PubMed  Google Scholar 

  45. Beattie SG, Goetzman E, Conlon T, Germain S, Walter G, Campbell-Thompson M et al. Biochemical correction of short-chain acyl-coenzyme A dehydrogenase after portal vein injection of rAAV8-SCAD. Hum Gene Ther 2008; 19: 579–588.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  46. Samulski RJ, Berns KI, Tan M, Muzyczka N . Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc Natl Acad Sci USA 1982; 79: 2077–2081.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. Dandapat A, Hu CP, Chen J, Liu Y, Khan JA, Remeo F et al. Over-expression of angiotensin II type 2 receptor (agtr2) decreases collagen accumulation in atherosclerotic plaque. Biochem Biophys Res Commun 2008; 366: 871–877.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work is supported by the Grant R01 CA104873 from the NIH, a VA Merit Review grant and intramural funding support from the UAMS College of Medicine Research Council to PLH. We thank Dr Jurgen Kleinschmidt (Hiedelberg, Germany), Dr James Trempe (Toledo, OH), Dr Sergei Zolotukin (Gainesville, FL) and Dr Nicholas Muzyczka (Gainesville, FL) for their kind donations of pKEX-Rep78, pSH3, pDG8 and pSM620, respectively. We also thank Dr Philip Palade (Little Rock, AR) for reviewing this manuscript.

Author information

Authors and Affiliations

  1. Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    M Cao, H Zhu, S Bandyopadhyay, H You & P L Hermonat

  2. Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    M Cao, H Zhu, S Bandyopadhyay, H You & P L Hermonat

  3. Department of Research, Central Arkansas Veterans Healthcare System, Little Rock, AR, USA

    M Cao, H Zhu & P L Hermonat

  4. Division of Cardiology, University of Arkansas for Medical Sciences and VA Medical Center, Little Rock, AR, USA

    H Zhu

Authors
  1. M Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Bandyopadhyay
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H You
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P L Hermonat
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P L Hermonat.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cao, M., Zhu, H., Bandyopadhyay, S. et al. HPV-16 E1, E2 and E6 each complement the Ad5 helper gene set, increasing rAAV2 and wt AAV2 production. Gene Ther 19, 418–424 (2012). https://doi.org/10.1038/gt.2011.115

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2011.115

Keywords

Search

Advanced search

Quick links