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.

  • Article
  • Published:

Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

Nature Chemistry volume 6, pages 28–33 (2014)Cite this article

Subjects

Abstract

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on–off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

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: Force-dependent difference in length between DNA and a saturated A3G–DNA complex allows us to measure A3G binding.
Figure 2: Single-molecule method to measure fast and slow fractions of A3G binding.
Figure 3: Quantifying A3G binding reveals association and dissociation rates for fast and slow binding modes.
Figure 4: Oligomerization-defective mutant F126A/W127A (FW) demonstrates that the slow kinetics observed for wild-type A3G is due to oligomerization.
Figure 5: Models for A3G oligomerization in vitro and in virio.

Similar content being viewed by others

References

  1. Malim, M. H. APOBEC proteins and intrinsic resistance to HIV-1 infection. Philos. Trans. R. Soc. Lond. B 364, 675–687 (2009).

    Article  CAS  Google Scholar 

  2. Harris, R. S. & Liddament, M. T. Retroviral restriction by APOBEC proteins. Nature Rev. Immunol. 4, 868–877 (2004).

    Article  CAS  Google Scholar 

  3. Duggal, N. K. & Emerman, M. Evolutionary conflicts between viruses and restriction factors shape immunity. Nature Rev. Immunol. 12, 687–695 (2012).

    Article  CAS  Google Scholar 

  4. Chiu, Y. L. & Greene, W. C. The APOBEC3 cytidine deaminases: an innate defensive network opposing exogenous retroviruses and endogenous retroelements. Annu. Rev. Immunol. 26, 317–353 (2008).

    Article  CAS  Google Scholar 

  5. Sheehy, A. M., Gaddis, N. C., Choi, J. D. & Malim, M. H. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418, 646–650 (2002).

    Article  CAS  Google Scholar 

  6. Holmes, R. K., Malim, M. H. & Bishop, K. N. APOBEC-mediated viral restriction: not simply editing? Trends Biochem. Sci. 32, 118–128 (2007).

    Article  CAS  Google Scholar 

  7. Fisher, A. G. et al. The sor gene of HIV-1 is required for efficient virus transmission in vitro. Science 237, 888–893 (1987).

    Article  CAS  Google Scholar 

  8. Strebel, K. et al. The HIV ‘A’ (sor) gene product is essential for virus infectivity. Nature 328, 728–730 (1987).

    Article  CAS  Google Scholar 

  9. Goila-Gaur, R. & Strebel, K. HIV-1 Vif, APOBEC, and intrinsic immunity. Retrovirology 5, 1–16 (2008).

    Article  Google Scholar 

  10. Lecossier, D., Bouchonnet, F., Clavel, F. & Hance, A. J. Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science 300, 1112 (2003).

    Article  CAS  Google Scholar 

  11. Mangeat, B. et al. Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424, 99–103 (2003).

    Article  CAS  Google Scholar 

  12. Zhang, H. et al. The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424, 94–98 (2003).

    Article  CAS  Google Scholar 

  13. Suspène, R. et al. APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase. Nucleic Acids Res. 32, 2421–2429 (2004).

    Article  Google Scholar 

  14. Yu, Q. et al. Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome. Nature Struct. Mol. Biol. 11, 435–442 (2004).

    Article  CAS  Google Scholar 

  15. Harris, R. S. et al. DNA deamination mediates innate immunity to retroviral infection. Cell 113, 803–809 (2003); erratum 116, 629 (2004).

    Article  CAS  Google Scholar 

  16. Levin, J. G., Mitra, M., Mascarenhas, A. & Musier-Forsyth, K. Role of HIV-1 nucleocapsid protein in HIV-1 reverse transcription. RNA Biol. 7, 754–774 (2010).

    Article  CAS  Google Scholar 

  17. Newman, E. N. C. et al. Antiviral function of APOBEC3G can be dissociated from cytidine deaminase activity. Curr. Biol. 15, 166–170 (2005).

    Article  CAS  Google Scholar 

  18. Holmes, R. K., Koning, F. A., Bishop, K. N. & Malim, M. H. APOBEC3F can inhibit the accumulation of HIV-1 reverse transcription products in the absence of hypermutation—comparisons with APOBEC3G. J. Biol. Chem. 282, 2587–2595 (2007).

    Article  CAS  Google Scholar 

  19. Iwatani, Y., Takeuchi, H., Strebel, K. & Levin, J. G. Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect. J. Virol. 80, 5992–6002 (2006).

    Article  CAS  Google Scholar 

  20. Luo, K. et al. Cytidine deaminases APOBEC3G and APOBEC3F interact with human immunodeficiency virus type 1 integrase and inhibit proviral DNA formation. J .Virol. 81, 7238–7248 (2007).

    Article  CAS  Google Scholar 

  21. Turelli, P., Mangeat, B., Jost, S., Vianin, S. & Trono, D. Inhibition of hepatitis B virus replication by APOBEC3G. Science 303, 1829 (2004).

    Article  Google Scholar 

  22. Bogerd, H. P., Wiegand, H. L., Doehle, B. P., Lueders, K. K. & Cullen, B. R. APOBEC3A and APOBEC3B are potent inhibitors of LTR-retrotransposon function in human cells. Nucleic Acids Res. 34, 89–95 (2006).

    Article  CAS  Google Scholar 

  23. Bogerd, H. P. et al. Cellular inhibitors of long interspersed element 1 and Alu retrotransposition. Proc. Natl Acad. Sci. USA 103, 8780–8785 (2006).

    Article  CAS  Google Scholar 

  24. Chen, H. et al. APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons. Curr. Biol. 16, 480–485 (2006).

    Article  CAS  Google Scholar 

  25. Muckenfuss, H. et al. APOBEC3 proteins inhibit human LINE-1 retrotransposition. J. Biol. Chem. 281, 22161–22172 (2006).

    Article  CAS  Google Scholar 

  26. Kinomoto, M. et al. All APOBEC3 family proteins differentially inhibit LINE-1 retrotransposition. Nucleic Acids Res. 35, 2955–2964 (2007).

    Article  CAS  Google Scholar 

  27. Niewiadomska, A. M. et al. Differential inhibition of long interspersed element 1 by APOBEC3 does not correlate with high-molecular-mass-complex formation or P-body association. J. Virol. 81, 9577–9583 (2007).

    Article  CAS  Google Scholar 

  28. Bulliard, Y. et al. Structure–function analyses point to a polynucleotide-accommodating groove essential for APOBEC3A restriction activities. J .Virol. 85, 1765–1776 (2011).

    Article  CAS  Google Scholar 

  29. Narvaiza, I. et al. Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase. PLoS Pathog. 5, e1000439 (2009).

    Article  Google Scholar 

  30. Bishop, K. N., Verma, M., Kim, E. Y., Wolinsky, S. M. & Malim, M. H. APOBEC3G inhibits elongation of HIV-1 reverse transcripts. PLoS Pathog. 4, e1000231 (2008).

    Article  Google Scholar 

  31. Adolph, M. B., Webb, J. & Chelico, L. Retroviral restriction factor APOBEC3G delays the initiation of DNA synthesis by HIV-1 reverse transcriptase. Plos One 8, e64196 (2013).

    Article  CAS  Google Scholar 

  32. Iwatani, Y. et al. Deaminase-independent inhibition of HIV-1 reverse transcription by APOBEC3G. Nucleic Acids Res. 35, 7096–7108 (2007).

    Article  CAS  Google Scholar 

  33. Li, X. Y., Guo, F., Zhang, L. & Kleiman, L. & Cen S. APOBEC3G inhibits DNA strand transfer during HIV-1 reverse transcription. J. Biol. Chem. 282, 32065–32074 (2007).

    Article  CAS  Google Scholar 

  34. Mbisa, J. L. et al. Human immunodeficiency virus type 1 cDNAs produced in the presence of APOBEC3G exhibit defects in plus-strand DNA transfer and integration. J. Virol. 81, 7099–7110 (2007).

    Article  CAS  Google Scholar 

  35. Gillick, K. et al. Suppression of HIV-1 infection by APOBEC3 proteins in primary human CD4+ T cells is associated with inhibition of processive reverse transcription as well as excessive cytidine deamination. J. Virol. 87, 1508–1517 (2013).

    Article  CAS  Google Scholar 

  36. Xu, H. Z. et al. Stoichiometry of the antiviral protein APOBEC3G in HIV-1 virions. Virology 360, 247–256 (2007).

    Article  CAS  Google Scholar 

  37. Chelico, L., Sacho, E. J., Erie, D. A. & Goodman, M. F. A model for oligomeric regulation of APOBEC3G cytosine deaminase-dependent restriction of HIV. J. Biol. Chem. 283, 13780–13791 (2008).

    Article  CAS  Google Scholar 

  38. Nowarski, R., Britan-Rosich, E., Shiloach, T. & Kotler, M. Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase. Nature Struct. Mol. Biol. 15, 1059–1066 (2008).

    Article  CAS  Google Scholar 

  39. King, G. A. et al. Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy. Proc. Natl Acad. Sci. USA 110, 3859–3864 (2013).

    Article  CAS  Google Scholar 

  40. Chaurasiya, K. R., Paramanathan, T., McCauley, M. J. & Williams, M. C. Biophysical characterization of DNA binding from single molecule force measurements. Phys. Life Rev. 7, 299–341 (2010).

    Article  Google Scholar 

  41. Senavirathne, G. et al. Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution. J. Biol. Chem. 287, 15826–15835 (2012).

    Article  CAS  Google Scholar 

  42. Chelico, L., Prochnow, C., Erie, D. A., Chen, X. S. & Goodman, M. F. Structural model for deoxycytidine deamination mechanisms of the HIV-1 inactivation enzyme APOBEC3G. J. Biol. Chem. 285, 16195–16205 (2010).

    Article  CAS  Google Scholar 

  43. Huthoff, H., Autore, F., Gallois-Montbrun, S., Fraternali, F. & Malim, M. H. RNA-dependent oligomerization of APOBEC3G is required for restriction of HIV-1. PLoS Pathog. 5, e1000330 (2009).

    Article  Google Scholar 

  44. Bélanger, K., Savoie, M., Rosales Gerpe, M. C., Couture, J-F. & Langlois, M-A. Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses. Nucleic Acids Res. 41, 7438–7452 (2013).

    Article  Google Scholar 

  45. Soros, V. & Greene, W. APOBEC3G and HIV-1: strike and counterstrike. Curr. HIV/AIDS Rep. 4, 3–9 (2007).

    Article  Google Scholar 

  46. Wang, X. X. et al. The cellular antiviral protein APOBEC3G interacts with HIV-1 reverse transcriptase and inhibits its function during viral replication. J. Virol. 86, 3777–3786 (2012).

    Article  CAS  Google Scholar 

  47. Coffin, J. M., Hughes, S. H. & Varmus, H. E. Retroviruses (Cold Spring Harbor Laboratory Press, 1997).

    Google Scholar 

  48. Vo, M. N., Barany, G., Rouzina, I. & Musier-Forsyth, K. Mechanistic studies of mini-TAR RNA/DNA annealing in the absence and presence of HIV-1 nucleocapsid protein. J. Mol. Biol. 363, 244–261 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank D. Pollpeter, M.H. Malim and D. Rueda for valuable discussions, and M.F. Goodman for his generous gift of the F126A/W127A mutant clone. This work was supported in part by the National Institutes of Health (GM072462 to M.C.W. and GM065056 to K.M-F.) and the National Science Foundation (MCB-1243883 to M.C.W.), the Japan Society for the Promotion of Science (JSPS; KAKENHI_24590568 to Y.I.), and in part by funds from the NIH Intramural Research Program (NICHD; to J.G.L.). K.R.C. was supported by the NSF IGERT Program (DGE-0504331).

Author information

Authors and Affiliations

  1. Department of Physics, Northeastern University, Boston, 02115, Massachusetts, USA

    Kathy R. Chaurasiya, Micah J. McCauley, Hylkje Geertsema & Mark C. Williams

  2. Department of Chemistry and Biochemistry, Center for Retrovirus Research, and Center for RNA Biology, The Ohio State University, Columbus, 43210, Ohio, USA

    Wei Wang, Dominic F. Qualley & Karin Musier-Forsyth

  3. Section on Viral Gene Regulation, Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Tiyun Wu, Amber Hertz, Yasumasa Iwatani & Judith G. Levin

  4. Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, 460-0001, Aichi, Japan

    Shingo Kitamura & Yasumasa Iwatani

  5. Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, 15260, Pennsylvania, USA

    Denise S. B. Chan

  6. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, 55455, Minnesota, USA

    Ioulia Rouzina

Authors
  1. Kathy R. Chaurasiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Micah J. McCauley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dominic F. Qualley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tiyun Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shingo Kitamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hylkje Geertsema
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Denise S. B. Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Amber Hertz
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yasumasa Iwatani
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Judith G. Levin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Karin Musier-Forsyth
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Ioulia Rouzina
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Mark C. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.C.W., K.R.C. and I.R. designed the experiments. K.R.C. performed experiments and analysed the data. M.M. performed experiments with the mutant. H.G. performed preliminary experiments. S.K., W.W., D.F.Q., T.W., Y.I., D.S.B.C. and A.H. prepared the proteins. I.R. developed the binding model. K.R.C., M.C.W., I.R., J.G.L. and K.M-F. wrote the manuscript.

Corresponding author

Correspondence to Mark C. Williams.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 429 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chaurasiya, K., McCauley, M., Wang, W. et al. Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein. Nature Chem 6, 28–33 (2014). https://doi.org/10.1038/nchem.1795

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.1795

This article is cited by

Search

Advanced 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