Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1

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

  2. 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).

  3. 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).

  4. 4

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

  5. 5

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

  6. 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).

  7. 7

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

  8. 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).

  9. 9

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

  10. 10

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

  11. 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).

  12. 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).

  13. 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).

  14. 14

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

  15. 15

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

  16. 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).

  17. 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).

  18. 18

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

  19. 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).

  20. 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).

  21. 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).

  22. 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).

  23. 23

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

  24. 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).

  25. 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).

  26. 26

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

  27. 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).

  28. 28

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

  29. 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).

  30. 30

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

  31. 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).

  32. 32

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

  33. 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).

  34. 34

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

  35. 35

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

  36. 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).

  37. 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).

  38. 38

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

  39. 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).

  40. 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).

  41. 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).

  42. 42

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

  43. 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).

  44. 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).

  45. 45

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

  46. 46

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

  47. 47

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

  48. 48

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

  49. 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).

  50. 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).

  51. 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).

  52. 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).

  53. 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).

  54. 54

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

  55. 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).

  56. 56

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

  57. 57

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

  58. 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).

  59. 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).

  60. 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).

  61. 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).

  62. 62

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

Download references

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).

Author information

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.

Correspondence to Moshe Kotler.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Table 1 (PDF 564 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading