Hypoxia-induced human deoxyribonuclease I is a cellular restriction factor of hepatitis B virus

Abstract

Numerous human APOBEC3 cytidine deaminases have proven to be, inter alia, host cell restriction factors for retroviruses and hepadnaviruses. Although they can bind to genomic RNA and become encapsidated, they are only catalytically active on single-stranded DNA. As there are many cellular deoxyribonucleases (DNases), we hypothesized that a parallel could be struck between APOBEC3 and DNases. For human hepatitis B virus (HBV), we show that DNase I can considerably reduce the virion genome copy number from a variety of transfected or infected cells. DNASE1 is overexpressed and encapsidated in HBV particles in vitro in hypoxic environments and in vivo in cirrhotic patient livers as well as in the serum of infected patients. The use of CoCl2 and dimethyloxalylglycine, mimetic agents used to induce hypoxia by inhibiting prolyl hydroxylase enzymes that stabilize hypoxia-inducible factor (HIF)-1α, showed that the formation of HIF-1α/HIF-1β heterodimers results in the induction of DNASE1. Indeed, transfection with HIF-1α and HIF-1β expression constructs upregulated DNASE1. These findings suggest that human DNase I can impact HBV replication through the catabolism of the DNA genome within the capsid. The activity of DNases in general may explain in part the high frequency of empty or ‘light’ hepatitis B virions observed in vivo.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: DNase I constructs and activity.
Fig. 2: DNase I reduces HBV DNA levels.
Fig. 3: DNase I is incorporated within HBV particles.
Fig. 4: Hypoxia enhances DNASE1 expression and restricts HBV.
Fig. 5: Expression analysis of DNASE1 and HBV restriction in CoCl2-treated HepG2 cells.
Fig. 6: DNASE1 upregulation in vivo and HIF-1α/HIF-1β involvement.

Data availability

The data that support the findings of this study are available from the corresponding authors on request.

References

  1. 1.

    Malim, M. H. & Bieniasz, P. D. HIV restriction factors and mechanisms of evasion. Cold Spring Harb. Perspect. Med. 2, a006940 (2012).

    Article  Google Scholar 

  2. 2.

    Simon, V., Bloch, N. & Landau, N. R. Intrinsic host restrictions to HIV-1 and mechanisms of viral escape. Nat. Immunol. 16, 546–553 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Bishop, K. N. et al. Cytidine deamination of retroviral DNA by diverse APOBEC proteins. Curr. Biol. 14, 1392–1396 (2004).

    CAS  Article  Google Scholar 

  4. 4.

    Kirchhoff, F. Immune evasion and counteraction of restriction factors by HIV-1 and other primate lentiviruses. Cell Host Microbe 8, 55–67 (2010).

    CAS  Article  Google Scholar 

  5. 5.

    Hatziioannou, T., Perez-Caballero, D., Yang, A., Cowan, S. & Bieniasz, P. D. Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5α. Proc. Natl Acad. Sci. USA 101, 10774–10779 (2004).

    CAS  Article  Google Scholar 

  6. 6.

    Laguette, N. et al. Samhd1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature 474, 654–657 (2011).

    CAS  Article  Google Scholar 

  7. 7.

    Neil, S. J., Zang, T. & Bieniasz, P. D. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451, 425–430 (2008).

    CAS  Article  Google Scholar 

  8. 8.

    Rosa, A. et al. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature 526, 212–217 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Stremlau, M. et al. The cytoplasmic body component TRIM5α restricts HIV-1 infection in Old World monkeys. Nature 427, 848–853 (2004).

    CAS  Article  Google Scholar 

  10. 10.

    Usami, Y., Wu, Y. & Gottlinger, H. G. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef. Nature 526, 218–223 (2015).

    CAS  Article  Google Scholar 

  11. 11.

    Suspène, R. et al. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proc. Natl Acad. Sci. USA 102, 8321–8326 (2005).

    Article  Google Scholar 

  12. 12.

    Ko, C., Lee, S., Windisch, M. P. & Ryu, W. S. DDX3 DEAD-box RNA helicase is a host factor that restricts hepatitis B virus replication at the transcriptional level. J. Virol. 88, 13689–13698 (2014).

    Article  Google Scholar 

  13. 13.

    Chelico, L., Pham, P., Calabrese, P. & Goodman, M. F. APOBEC3G DNA deaminase acts processively 3′→5′ on single-stranded DNA. Nat. Struct. Mol. Biol. 13, 392–399 (2006).

    CAS  Article  Google Scholar 

  14. 14.

    Peitsch, M. C. et al. Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J. 12, 371–377 (1993).

    CAS  Article  Google Scholar 

  15. 15.

    Kimura-Kataoka, K. et al. Identification of the functional alleles of the nonsynonymous single-nucleotide polymorphisms potentially implicated in systemic lupus erythematosus in the human deoxyribonuclease I gene. DNA Cell Biol. 33, 492–502 (2014).

    CAS  Article  Google Scholar 

  16. 16.

    Oliveri, M. et al. DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis. Eur. J. Immunol. 31, 743–751 (2001).

    CAS  Article  Google Scholar 

  17. 17.

    Amoura, Z. et al. Circulating plasma levels of nucleosomes in patients with systemic lupus erythematosus: correlation with serum antinucleosome antibody titers and absence of clear association with disease activity. Arthritis Rheum. 40, 2217–2225 (1997).

    CAS  Article  Google Scholar 

  18. 18.

    Alberti, A., Diana, S., Scullard, G. H., Eddleston, W. F. & Williams, R. Full and empty Dane particles in chronic hepatitis B virus infection: relation to hepatitis B e antigen and presence of liver damage. Gastroenterology 75, 869–874 (1978).

    CAS  Article  Google Scholar 

  19. 19.

    Kaplan, P. M., Ford, E. C., Purcell, R. H. & Gerin, J. L. Demonstration of subpopulations of Dane particles. J. Virol. 17, 885–893 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Luckenbaugh, L., Kitrinos, K. M., Delaney, W. E. T. & Hu, J. Genome-free hepatitis B virion levels in patient sera as a potential marker to monitor response to antiviral therapy. J. Viral Hepat. 22, 561–570 (2015).

    CAS  Article  Google Scholar 

  21. 21.

    Ning, X. et al. Secretion of genome-free hepatitis B virus—single strand blocking model for virion morphogenesis of para-retrovirus. PLoS Pathog. 7, e1002255 (2011).

    CAS  Article  Google Scholar 

  22. 22.

    Gerin, J. L., Ford, E. C. & Purcell, R. H. Biochemical characterization of Australia antigen. Evidence for defective particles of hepatitis B virus. Am. J. Pathol. 81, 651–668 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Sakamoto, Y. et al. Full and empty particles of hepatitis B virus in hepatocytes from patients with HBsAg-positive chronic active hepatitis. Lab. Invest. 48, 678–682 (1983).

    CAS  PubMed  Google Scholar 

  24. 24.

    Wang, G. L., Jiang, B. H., Rue, E. A. & Semenza, G. L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl Acad. Sci. USA 92, 5510–5514 (1995).

    CAS  Article  Google Scholar 

  25. 25.

    Fu, X. S., Choi, E., Bubley, G. J. & Balk, S. P. Identification of hypoxia-inducible factor-1α (HIF-1α) polymorphism as a mutation in prostate cancer that prevents normoxia-induced degradation. Prostate 63, 215–221 (2005).

    CAS  Article  Google Scholar 

  26. 26.

    Zhang, C. et al. Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA. Proc. Natl Acad. Sci. USA 113, 2047–2056 (2016).

    Article  Google Scholar 

  27. 27.

    Zhang, H. et al. HIF-1α activates hypoxia-induced PFKFB4 expression in human bladder cancer cells. Biochem. Biophys. Res. Commun. 476, 146–152 (2016).

    CAS  Article  Google Scholar 

  28. 28.

    Zhu, B. et al. DNase I aggravates islet β-cell apoptosis in type 2 diabetes. Mol. Med. Rep. 13, 4577–4584 (2016).

    CAS  Article  Google Scholar 

  29. 29.

    Kominato, Y. et al. Hypoxia induces upregulation of the deoxyribonuclease I gene in the human pancreatic cancer cell line QGP-1. Biochim. Biophys. Acta 1770, 1567–1575 (2007).

    CAS  Article  Google Scholar 

  30. 30.

    Liaw, Y. F. & Chu, C. M. Hepatitis B virus infection. Lancet 373, 582–592 (2009).

    CAS  Article  Google Scholar 

  31. 31.

    Parsiegla, G., Noguere, C., Santell, L., Lazarus, R. A. & Bourne, Y. The structure of human DNase I bound to magnesium and phosphate ions points to a catalytic mechanism common to members of the DNase I-like superfamily. Biochemistry 51, 10250–10258 (2012).

    CAS  Article  Google Scholar 

  32. 32.

    Stenglein, M. D., Burns, M. B., Li, M., Lengyel, J. & Harris, R. S. APOBEC3 proteins mediate the clearance of foreign DNA from human cells. Nat. Struct. Mol. Biol. 17, 222–229 (2010).

    CAS  Article  Google Scholar 

  33. 33.

    Ladner, S. K. et al. Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob. Agents Chemother. 41, 1715–1720 (1997).

    CAS  Article  Google Scholar 

  34. 34.

    Suspène, R., Henry, M., Guillot, S., Wain-Hobson, S. & Vartanian, J. P. Recovery of APOBEC3-edited human immunodeficiency virus G->A hypermutants by differential DNA denaturation PCR. J. Gen. Virol. 86, 125–129 (2005).

    Article  Google Scholar 

  35. 35.

    Vartanian, J. P. et al. Massive APOBEC3 editing of hepatitis B viral DNA in cirrhosis. PLoS Pathog. 6, e1000928 (2010).

    Article  Google Scholar 

  36. 36.

    Bayard, F. et al. T-cell responses to hepatitis B splice-generated protein of hepatitis B virus and inflammatory cytokines/chemokines in chronic hepatitis B patients. ANRS study: HB EP 02 HBSP-FIBRO. J. Viral Hepat. 19, 872–880 (2012).

    CAS  Article  Google Scholar 

  37. 37.

    Couillin, I. et al. Specific vaccine therapy in chronic hepatitis B: induction of T cell proliferative responses specific for envelope antigens. J. Infect. Dis. 180, 15–26 (1999).

    CAS  Article  Google Scholar 

  38. 38.

    Ke, Q. & Costa, M. Hypoxia-inducible factor-1 (HIF-1). Mol. Pharmacol. 70, 1469–1480 (2006).

    CAS  Article  Google Scholar 

  39. 39.

    Semenza, G. L. Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis. Blood 114, 2015–2019 (2009).

    CAS  Article  Google Scholar 

  40. 40.

    Sonna, L. A., Cullivan, M. L., Sheldon, H. K., Pratt, R. E. & Lilly, C. M. Effect of hypoxia on gene expression by human hepatocytes (HepG2). Physiol. Genomics 12, 195–207 (2003).

    CAS  Article  Google Scholar 

  41. 41.

    Henry, M. et al. Genetic editing of HBV DNA by monodomain human APOBEC3 cytidine deaminases and the recombinant nature of APOBEC3G. PLoS ONE 4, e4277 (2009).

    Article  Google Scholar 

  42. 42.

    Sasaki, Y., Miyoshi, D. & Sugimoto, N. Regulation of DNA nucleases by molecular crowding. Nucleic Acids Res. 35, 4086–4093 (2007).

    CAS  Article  Google Scholar 

  43. 43.

    Gibson, W. Structure and formation of the cytomegalovirus virion. Curr. Top. Microbiol. Immunol. 325, 187–204 (2008).

    CAS  PubMed  Google Scholar 

  44. 44.

    Tandon, R. & Mocarski, E. S. Viral and host control of cytomegalovirus maturation. Trends Microbiol. 20, 392–401 (2012).

    CAS  Article  Google Scholar 

  45. 45.

    Suspène, R. et al. Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism. Proc. Natl Acad. Sci. USA 108, 4858–4863 (2011).

    Article  Google Scholar 

  46. 46.

    Aynaud, M. M. et al. Human Tribbles 3 protects nuclear DNA from cytidine deamination by APOBEC3A. J. Biol. Chem. 287, 39182–39192 (2012).

    CAS  Article  Google Scholar 

  47. 47.

    Zahn, A. & Allain, J. P. Hepatitis C virus and hepatitis B virus bind to heparin: purification of largely IgG-free virions from infected plasma by heparin chromatography. J. Gen. Virol. 86, 677–685 (2005).

    CAS  Article  Google Scholar 

  48. 48.

    Bouzidi, M. S. et al. APOBEC3DE antagonizes hepatitis B virus restriction factors APOBEC3F and APOBEC3G. J. Mol. Biol. 428, 3514–3528 (2016).

    CAS  Article  Google Scholar 

  49. 49.

    De Castro Martin, I. F. et al. Influenza virus genome reaches the plasma membrane via a modified endoplasmic reticulum and Rab11-dependent vesicles. Nat. Commun. 8, 1396 (2017).

    Article  Google Scholar 

  50. 50.

    Slot, J. W., Geuze, H. J., Gigengack, S., Lienhard, G. E. & James, D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J. Cell Biol. 113, 123–135 (1991).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Lanzhou Institute of Biological Products Co., Ltd. is a subsidiary company of China National Biotec Group Company Limited. This work was supported by grants from the Institut Pasteur and Centre National de la Recherche Scientifique. C.H. and M.S.B. were supported by the Allocation de Recherche du Ministère de la Recherche and Ligue contre le Cancer, respectively. X.L. was supported by a stipend from the Lanzhou Institute of Biological Products Co., Ltd, subsidiary company of China National Biotec Group Company Limited and by a stipend from the Pasteur-Paris University International PhD program. V.L. was supported by the Fondation pour la Recherche Medicale (project no. ING20160435328). C.D. was supported by an EMBO Long Term Fellowship (EMBO ALTF 1428–2016). The study of patients was sponsored by the French National Agency for research on AIDS and hepatitis (ANRS). This work has been published within the framework of IdEx Université de Strasbourg and has received funding from the French State via the French National Research Agency (ANR) as part of the program ‘Investissements d’avenir’ to R.G. This work was supported by an ATIP-AVENIR starting grant to R.G. We would like to thank S. Urban for providing HepG2-NTCP cells; P. Pineau and A. Marchio for the cirrhotic liver samples; M.-L. Michel and S. Pol for serum samples; C. Neuveut, P. Moreau, P. Maillard and B. Quioc for HBV infection; Y. Jacob for the HIF-1α and HIF-1β plasmids; J. Krijnse-Locker and C. Schmitt for electron microscopy immunolabelling and V. Caval, N. Frampton and J. McKeating for helpful discussions.

Author information

Affiliations

Authors

Contributions

S.W.-H. and J.-P.V. designed the research. C.H., X.X.L., R.S., V.T., M.S.B., V.L., C.D. and R.G. performed the experiments. C.H., X.X.L., R.S., V.T., M.S.B., V.L., C.D., S.W.-H., R.G. and J.-P.V. analysed the data. S.W.-H., R.G. and J.-P.V. wrote the paper.

Corresponding authors

Correspondence to Raphaël Gaudin or Jean-Pierre Vartanian.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–7, Supplementary Tables 1 and 2, Raw Image Figures and Raw Image.

Reporting Summary

Supplementary Video 1

Three-dimensional time-lapse of the crop presented in Supplementary Figure 5.

Supplementary Video 2

Three-dimensional isosurface time-lapse of the of the crop presented in Supplementary Figure 5.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hallez, C., Li, X., Suspène, R. et al. Hypoxia-induced human deoxyribonuclease I is a cellular restriction factor of hepatitis B virus. Nat Microbiol 4, 1196–1207 (2019). https://doi.org/10.1038/s41564-019-0405-x

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing