Letter | Published:

CG dinucleotide suppression enables antiviral defence targeting non-self RNA

Nature volume 550, pages 124127 (05 October 2017) | Download Citation

Abstract

Vertebrate genomes exhibit marked CG suppression—that is, lower than expected numbers of 5′-CG-3′ dinucleotides1. This feature is likely to be due to C-to-T mutations that have accumulated over hundreds of millions of years, driven by CG-specific DNA methyl transferases and spontaneous methyl-cytosine deamination. Many RNA viruses of vertebrates that are not substrates for DNA methyl transferases mimic the CG suppression of their hosts2,3,4. This property of viral genomes is unexplained4,5,6. Here we show, using synonymous mutagenesis, that CG suppression is essential for HIV-1 replication. The deleterious effect of CG dinucleotides on HIV-1 replication was cumulative, associated with cytoplasmic RNA depletion, and was exerted by CG dinucleotides in both translated and non-translated exonic RNA sequences. A focused screen using small inhibitory RNAs revealed that zinc-finger antiviral protein (ZAP)7 inhibited virion production by cells infected with CG-enriched HIV-1. Crucially, HIV-1 mutants containing segments whose CG content mimicked random nucleotide sequence were defective in unmanipulated cells, but replicated normally in ZAP-deficient cells. Crosslinking–immunoprecipitation–sequencing assays demonstrated that ZAP binds directly and selectively to RNA sequences containing CG dinucleotides. These findings suggest that ZAP exploits host CG suppression to identify non-self RNA. The dinucleotide composition of HIV-1, and perhaps other RNA viruses, appears to have adapted to evade this host defence.

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References

  1. 1.

    & Compositional differences within and between eukaryotic genomes. Proc. Natl Acad. Sci. USA 94, 10227–10232 (1997)

  2. 2.

    , & Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? J. Virol. 68, 2889–2897 (1994)

  3. 3.

    & Dinucleotide and stop codon frequencies in single-stranded RNA viruses. J. Gen. Virol. 78, 2859–2870 (1997)

  4. 4.

    , , & Patterns of evolution and host gene mimicry in influenza and other RNA viruses. PLoS Pathog. 4, e1000079 (2008)

  5. 5.

    et al. CpG usage in RNA viruses: data and hypotheses. PLoS One 8, e74109 (2013)

  6. 6.

    et al. Reply to Simmonds et al.: Codon pair and dinucleotide bias have not been functionally distinguished. Proc. Natl Acad. Sci. USA 112, E3635–E3636 (2015)

  7. 7.

    , & Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein. Science 297, 1703–1706 (2002)

  8. 8.

    , & On the nucleotide composition and structure of retroviral RNA genomes. Virus Res. 193, 16–23 (2014)

  9. 9.

    & Transcriptional and posttranscriptional regulation of HIV-1 gene expression. Cold Spring Harb. Perspect. Med. 2, a006916 (2012)

  10. 10.

    et al. TRIM25 enhances the antiviral action of zinc-finger antiviral protein (ZAP). PLoS Pathog. 13, e1006145 (2017)

  11. 11.

    et al. TRIM25 is required for the antiviral activity of zinc finger antiviral protein. J. Virol. 91, e00088–17 (2017)

  12. 12.

    et al. Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation. Proc. Natl Acad. Sci. USA 108, 15834–15839 (2011)

  13. 13.

    , , , & The zinc finger antiviral protein directly binds to specific viral mRNAs through the CCCH zinc finger motifs. J. Virol. 78, 12781–12787 (2004)

  14. 14.

    & ZAP-mediated mRNA degradation. RNA Biol. 5, 65–67 (2008)

  15. 15.

    et al. Structure of N-terminal domain of ZAP indicates how a zinc-finger protein recognizes complex RNA. Nat. Struct. Mol. Biol. 19, 430–435 (2012)

  16. 16.

    , & Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA. Protein Cell 1, 752–759 (2010)

  17. 17.

    et al. Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J. Virol. 77, 11555–11562 (2003)

  18. 18.

    et al. Inhibition of filovirus replication by the zinc finger antiviral protein. J. Virol. 81, 2391–2400 (2007)

  19. 19.

    et al. Inhibition of hepatitis B virus replication by the host zinc finger antiviral protein. PLoS Pathog. 9, e1003494 (2013)

  20. 20.

    et al. Identification and characterization of alphavirus M1 as a selective oncolytic virus targeting ZAP-defective human cancers. Proc. Natl Acad. Sci. USA 111, E4504–E4512 (2014)

  21. 21.

    , , , & The broad-spectrum antiviral protein ZAP restricts human retrotransposition. PLoS Genet. 11, e1005252 (2015)

  22. 22.

    & The zinc-finger antiviral protein ZAP inhibits LINE and Alu retrotransposition. PLoS Genet. 11, e1005121 (2015)

  23. 23.

    , , & Battle between influenza A virus and a newly identified antiviral activity of the PARP-containing ZAPL protein. Proc. Natl Acad. Sci. USA 112, 14048–14053 (2015)

  24. 24.

    , & The short form of the zinc finger antiviral protein inhibits influenza A virus protein expression and is antagonized by the virus-encoded NS1. J. Virol. 91, e01909–16 (2017)

  25. 25.

    et al. Virus attenuation by genome-scale changes in codon pair bias. Science 320, 1784–1787 (2008)

  26. 26.

    , , , & RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. eLife 3, e04531 (2014)

  27. 27.

    & Codon pair bias is a direct consequence of dinucleotide bias. Cell Reports 14, 55–67 (2016)

  28. 28.

    , & PARP13 regulates cellular mRNA post-transcriptionally and functions as a pro-apoptotic factor by destabilizing TRAILR4 transcript. Nat. Commun. 5, 5362 (2014)

  29. 29.

    et al. Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis. Cell 159, 1096–1109 (2014)

  30. 30.

    et al. PARalyzer: definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol. 12, R79 (2011)

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Acknowledgements

We thank T. Kueck for primary lymphocytes and S. Giese for assistance with smFISH. This work was supported NIH grants R01AI50111 and P50GM103297 (to P.D.B.)

Author information

Affiliations

  1. Laboratory of Retrovirology, The Rockefeller University, New York, New York, USA

    • Matthew A. Takata
    • , Daniel Gonçalves-Carneiro
    • , Trinity M. Zang
    • , Steven J. Soll
    • , Ashley York
    • , Daniel Blanco-Melo
    •  & Paul D. Bieniasz
  2. Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA

    • Trinity M. Zang
    • , Steven J. Soll
    •  & Paul D. Bieniasz

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Contributions

M.A.T. performed all experiments unless otherwise stated and wrote the paper. D.G.-C. performed some of the luciferase reporter experiments and bioinformatic analyses. T.M.Z. performed smFISH experiments. A.Y. performed some of the CLIP experiments. D.B.-M. generated the mutant sequence in silico. S.J.S. constructed and characterized the 16 original mutant HIV-1 strains. P.D.B. conceived the study, supervised the work and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Paul D. Bieniasz.

Reviewer Information Nature thanks G. Towers and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Supplementary information

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    Supplementary Information

    This file contains uncropped Western blots used in Figures 2 and 3, and in Extended Data Figures 2, 5 and 6.

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    Reporting Summary

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    Supplementary Data 1

    A codon by codon list of the mutations made in segment L

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    Supplementary Data 2

    Alignment of WT and mutant EH segments (Fasta format).

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    Supplementary Data 3

    Alignment of WT and mutant L segments (Fasta format).

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DOI

https://doi.org/10.1038/nature24039

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