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Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase

Nature Structural & Molecular Biology volume 15, pages 1059–1066 (2008)Cite this article

Abstract

Deamination of cytidine residues in single-stranded DNA (ssDNA) is an important mechanism by which apolipoprotein B mRNA-editing, catalytic polypeptide-like (APOBEC) enzymes restrict endogenous and exogenous viruses. The dynamic process underlying APOBEC-induced hypermutation is not fully understood. Here we show that enzymatically active APOBEC3G can be detected in wild-type Vif(+) HIV-1 virions, albeit at low levels. In vitro studies showed that single enzyme-DNA encounters result in distributive deamination of adjacent cytidines. Nonlinear translocation of APOBEC3G, however, directed scattered deamination of numerous targets along the DNA. Increased ssDNA concentrations abolished enzyme processivity in the case of short, but not long, DNA substrates, emphasizing the key role of rapid intersegmental transfer in targeting the deaminase. Our data support a model by which APOBEC3G intersegmental transfer via monomeric binding to two ssDNA segments results in dispersed hypermutation of viral genomes.

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Figure 1: Models for A3G translocation during retroviral reverse transcription.
Figure 2: Enzymatically active A3G is incorporated into HIV-1 virions.
Figure 3: Correlation to facilitated diffusion.
Figure 4: A3G acts distributively on adjacent cytidines.
Figure 5: Segmental and molecular transfer of A3G.
Figure 6: DNA tethering by monomeric A3G.

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References

  1. Riggs, A.D., Bourgeois, S. & Cohn, M. The lac repressor-operator interaction. 3. Kinetic studies. J. Mol. Biol. 53, 401–417 (1970).

    Article  CAS  Google Scholar 

  2. Berg, O.G., Winter, R.B. & von Hippel, P.H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry 20, 6929–6948 (1981).

    Article  CAS  Google Scholar 

  3. Winter, R.B., Berg, O.G. & von Hippel, P.H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 3. The Escherichia coli lac repressor–operator interaction: kinetic measurements and conclusions. Biochemistry 20, 6961–6977 (1981).

    Article  CAS  Google Scholar 

  4. von Hippel, P.H. & Berg, O.G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989).

    CAS  PubMed  Google Scholar 

  5. von Hippel, P.H. Protein-DNA recognition: new perspectives and underlying themes. Science 263, 769–770 (1994).

    Article  CAS  Google Scholar 

  6. Stanford, N.P., Szczelkun, M.D., Marko, J.F. & Halford, S.E. One- and three-dimensional pathways for proteins to reach specific DNA sites. EMBO J. 19, 6546–6557 (2000).

    Article  CAS  Google Scholar 

  7. Houtsmuller, A.B. et al. Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284, 958–961 (1999).

    Article  CAS  Google Scholar 

  8. Lever, M.A., Th'ng, J.P., Sun, X. & Hendzel, M.J. Rapid exchange of histone H1.1 on chromatin in living human cells. Nature 408, 873–876 (2000).

    Article  CAS  Google Scholar 

  9. Misteli, T. Protein dynamics: implications for nuclear architecture and gene expression. Science 291, 843–847 (2001).

    Article  CAS  Google Scholar 

  10. Gorman, J. & Greene, E.C. Visualizing one-dimensional diffusion of proteins along DNA. Nat. Struct. Mol. Biol. 15, 768–774 (2008).

    Article  CAS  Google Scholar 

  11. Menetski, J.P. & Kowalczykowski, S.C. Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the transfer reaction. J. Biol. Chem. 262, 2085–2092 (1987).

    CAS  PubMed  Google Scholar 

  12. Kozlov, A.G. & Lohman, T.M. Kinetic mechanism of direct transfer of Escherichia coli SSB tetramers between single-stranded DNA molecules. Biochemistry 41, 11611–11627 (2002).

    Article  CAS  Google Scholar 

  13. Deibert, M., Grazulis, S., Sasnauskas, G., Siksnys, V. & Huber, R. Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA. Nat. Struct. Biol. 7, 792–799 (2000).

    Article  CAS  Google Scholar 

  14. Halford, S.E., Gowers, D.M. & Sessions, R.B. Two are better than one. Nat. Struct. Biol. 7, 705–707 (2000).

    Article  CAS  Google Scholar 

  15. Lewis, M. et al. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247–1254 (1996).

    Article  CAS  Google Scholar 

  16. Guo, F., Gopaul, D.N. & van Duyne, G.D. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389, 40–46 (1997).

    Article  CAS  Google Scholar 

  17. Jarmuz, A. et al. An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics 79, 285–296 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Izumi, T., Shirakawa, K. & Takaori-Kondo, A. Cytidine deaminases as a weapon against retroviruses and a new target for antiviral therapy. Mini Rev. Med. Chem. 8, 231–238 (2008).

    Article  CAS  Google Scholar 

  20. Vartanian, J.P., Guetard, D., Henry, M. & Wain-Hobson, S. Evidence for editing of human papillomavirus DNA by APOBEC3 in benign and precancerous lesions. Science 320, 230–233 (2008).

    Article  CAS  Google Scholar 

  21. Bhattacharya, S., Navaratnam, N., Morrison, J.R., Scott, J. & Taylor, W.R. Cytosine nucleoside/nucleotide deaminases and apolipoprotein B mRNA editing. Trends Biochem. Sci. 19, 105–106 (1994).

    Article  CAS  Google Scholar 

  22. Reizer, J., Buskirk, S., Bairoch, A., Reizer, A. & Saier, M.H., Jr. A novel zinc-binding motif found in two ubiquitous deaminase families. Protein Sci. 3, 853–856 (1994).

    Article  CAS  Google Scholar 

  23. Navarro, F. et al. Complementary function of the two catalytic domains of APOBEC3G. Virology 333, 374–386 (2005).

    Article  CAS  Google Scholar 

  24. Hache, G., Liddament, M.T. & Harris, R.S. The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain. J. Biol. Chem. 280, 10920–10924 (2005).

    Article  CAS  Google Scholar 

  25. 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 

  26. Harris, R.S. et al. DNA deamination mediates innate immunity to retroviral infection. Cell 113, 803–809 (2003).

    Article  CAS  Google Scholar 

  27. 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 

  28. 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 

  29. Marin, M., Rose, K.M., Kozak, S.L. & Kabat, D. HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat. Med. 9, 1398–1403 (2003).

    Article  CAS  Google Scholar 

  30. 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 

  31. Rosler, C., Kock, J., Malim, M.H., Blum, H.E. & von Weizsacker, F. Comment on “Inhibition of hepatitis B virus replication by APOBEC3G”. Science 305, 1403 author reply 1403 (2004).

    Article  Google Scholar 

  32. 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 

  33. Suspene, 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  CAS  Google Scholar 

  34. Kock, J. & Blum, H.E. Hypermutation of hepatitis B virus genomes by APOBEC3G, APOBEC3C and APOBEC3H. J. Gen. Virol. 89, 1184–1191 (2008).

    Article  Google Scholar 

  35. Esnault, C. et al. APOBEC3G cytidine deaminase inhibits retrotransposition of endogenous retroviruses. Nature 433, 430–433 (2005).

    Article  CAS  Google Scholar 

  36. Schumacher, A.J., Nissley, D.V. & Harris, R.S. APOBEC3G hypermutates genomic DNA and inhibits Ty1 retrotransposition in yeast. Proc. Natl. Acad. Sci. USA 102, 9854–9859 (2005).

    Article  CAS  Google Scholar 

  37. Dutko, J.A., Schafer, A., Kenny, A.E., Cullen, B.R. & Curcio, M.J. Inhibition of a yeast LTR retrotransposon by human APOBEC3 cytidine deaminases. Curr. Biol. 15, 661–666 (2005).

    Article  CAS  Google Scholar 

  38. Chiu, Y.L. et al. High-molecular-mass APOBEC3G complexes restrict Alu retrotransposition. Proc. Natl. Acad. Sci. USA 103, 15588–15593 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  40. Kao, S. et al. The human immunodeficiency virus type 1 Vif protein reduces intracellular expression and inhibits packaging of APOBEC3G (CEM15), a cellular inhibitor of virus infectivity. J. Virol. 77, 11398–11407 (2003).

    Article  CAS  Google Scholar 

  41. Stopak, K., de Noronha, C., Yonemoto, W. & Greene, W.C. HIV-1 Vif blocks the antiviral activity of APOBEC3G by impairing both its translation and intracellular stability. Mol. Cell 12, 591–601 (2003).

    Article  CAS  Google Scholar 

  42. Yu, X. et al. Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302, 1056–1060 (2003).

    Article  CAS  Google Scholar 

  43. Opi, S. et al. Human immunodeficiency virus type 1 Vif inhibits packaging and antiviral activity of a degradation-resistant APOBEC3G variant. J. Virol. 81, 8236–8246 (2007).

    Article  CAS  Google Scholar 

  44. Soros, V.B., Yonemoto, W. & Greene, W.C. Newly synthesized APOBEC3G is incorporated into HIV virions, inhibited by HIV RNA, and subsequently activated by RNase H. PLoS Pathog. 3, e15 (2007).

    Article  Google Scholar 

  45. John, M., Coffin, S.H.H. & Varmus, H.E. Retroviruses (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999).

    Google Scholar 

  46. Briggs, J.A. et al. The stoichiometry of Gag protein in HIV-1. Nat. Struct. Mol. Biol. 11, 672–675 (2004).

    Article  CAS  Google Scholar 

  47. Chiu, Y.L. et al. Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 435, 108–114 (2005).

    Article  CAS  Google Scholar 

  48. Fersht, A. Enzyme Structure and Mechanism (Freeman, New York, NY, 1985).

    Google Scholar 

  49. 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).

    Article  CAS  Google Scholar 

  50. 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 

  51. Wedekind, J.E. et al. Nanostructures of APOBEC3G support a hierarchical assembly model of high molecular mass ribonucleoprotein particles from dimeric subunits. J. Biol. Chem. 281, 38122–38126 (2006).

    Article  CAS  Google Scholar 

  52. Burnett, A. & Spearman, P. APOBEC3G multimers are recruited to the plasma membrane for packaging into human immunodeficiency virus type 1 virus-like particles in an RNA-dependent process requiring the NC basic linker. J. Virol. 81, 5000–5013 (2007).

    Article  CAS  Google Scholar 

  53. 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 

  54. Opi, S. et al. Monomeric APOBEC3G is catalytically active and has antiviral activity. J. Virol. 80, 4673–4682 (2006).

    Article  CAS  Google Scholar 

  55. Bittner, M., Burke, R.L. & Alberts, B.M. Purification of the T4 gene 32 protein free from detectable deoxyribonuclease activities. J. Biol. Chem. 254, 9565–9572 (1979).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  57. Mulder, L.C., Harari, A. & Simon, V. Cytidine deamination induced HIV-1 drug resistance. Proc. Natl. Acad. Sci. USA 105, 5501–5506 (2008).

    Article  CAS  Google Scholar 

  58. Pillai, S.K., Wong, J.K. & Barbour, J.D. Turning up the volume on mutational pressure: is more of a good thing always better? (a case study of HIV-1 Vif and APOBEC3). Retrovirology 5, 26 (2008).

    Article  Google Scholar 

  59. DeStefano, J.J. et al. Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled. J. Biol. Chem. 266, 7423–7431 (1991).

    CAS  PubMed  Google Scholar 

  60. Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P.J. & Bambara, R.A. Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H. Proc. Natl. Acad. Sci. USA 97, 11978–11983 (2000).

    Article  CAS  Google Scholar 

  61. Langlois, M.A., Beale, R.C., Conticello, S.G. & Neuberger, M.S. Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine deaminases provides insight into their DNA target site specificities. Nucleic Acids Res. 33, 1913–1923 (2005).

    Article  CAS  Google Scholar 

  62. Gooch, B.D. & Cullen, B.R. Functional domain organization of human APOBEC3G. Virology 379, 118–124 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was carried out in the Krueger Laboratory with the support of N. and L. Glick, and P. and M. Weiss. We thank K. Strebel (US National Institutes of Health (NIH), Bethesda, Maryland, USA) for providing pcDNA-APOBEC3G. We thank E. Pikarsky, R. Harris and H. Matsuo for constructive and helpful discussion, and S. Amir for editing this manuscript. Anti-APOBEC3G-C-terminal antibody was obtained from J. Lingappa through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. This work was supported in part by the Israel Ministry of Industry Trade & Labor via the Nofar program, the Israel Ministry of Health and the United States-Israel Binational Science Foundation (BSF).

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  1. Department of Pathology and the Lautenberg Center for General and Tumor Immunology, the Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel

    Roni Nowarski, Elena Britan-Rosich, Tamar Shiloach & Moshe Kotler

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  1. Roni Nowarski
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R.N. and M.K. designed the experiments and wrote the paper; R.N. performed the experiments; E.B.-R. and T.S. contributed reagents and provided assistance.

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Correspondence to Moshe Kotler.

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Nowarski, R., Britan-Rosich, E., Shiloach, T. et al. Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase. Nat Struct Mol Biol 15, 1059–1066 (2008). https://doi.org/10.1038/nsmb.1495

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