Viral and cellular N6-methyladenosine and N6,2′-O-dimethyladenosine epitranscriptomes in the KSHV life cycle

Abstract

N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am) modifications (m6A/m) of messenger RNA mediate diverse cellular functions. Oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) has latent and lytic replication phases that are essential for the development of KSHV-associated cancers. To date, the role of m6A/m in KSHV replication and tumorigenesis is unclear. Here, we provide mechanistic insights by examining the viral and cellular m6A/m epitranscriptomes during KSHV latent and lytic infection. KSHV transcripts contain abundant m6A/m modifications during latent and lytic replication, and these modifications are highly conserved among different cell types and infection systems. Knockdown of YTHDF2 enhanced lytic replication by impeding KSHV RNA degradation. YTHDF2 binds to viral transcripts and differentially mediates their stability. KSHV latent infection induces 5′ untranslated region (UTR) hypomethylation and 3′UTR hypermethylation of the cellular epitranscriptome, regulating oncogenic and epithelial-mesenchymal transition pathways. KSHV lytic replication induces dynamic reprogramming of epitranscriptome, regulating pathways that control lytic replication. These results reveal a critical role of m6A/m modifications in KSHV lifecycle and provide rich resources for future investigations.

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: KSHV m6A/m epitranscriptome during viral latent infection.
Fig. 2: KSHV m6A/m epitranscriptome during viral lytic replication.
Fig. 3: Silencing of YTHDF2 enhances KSHV lytic replication.
Fig. 4: Reprogramming of cellular m6A/m epitranscriptome during KSHV latency.
Fig. 5: Reprogramming of cellular m6A/m epitranscriptome during KSHV lytic replication.

References

  1. 1.

    Ye, F., Lei, X. & Gao, S. J. Mechanisms of Kaposi’s sarcoma-associated herpesvirus latency and reactivation. Adv. Virol. 2011, 193860 (2011).

  2. 2.

    Desrosiers, R., Friderici, K. & Rottman, F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc. Natl Acad. Sci. USA 71, 3971–3975 (1974).

    CAS  PubMed  Google Scholar 

  3. 3.

    Dominissini, D. et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485, 201–206 (2012).

    CAS  PubMed  Google Scholar 

  4. 4.

    Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Linder, B. et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Meth. 12, 767–772 (2015).

    CAS  Google Scholar 

  6. 6.

    Bokar, J. A., Rath-Shambaugh, M. E., Ludwiczak, R., Narayan, P. & Rottman, F. Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex. J. Biol. Chem. 269, 17697–17704 (1994).

    CAS  PubMed  Google Scholar 

  7. 7.

    Bokar, J. A., Shambaugh, M. E., Polayes, D., Matera, A. G. & Rottman, F. M. Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase. RNA 3, 1233–1247 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Schwartz, S. et al. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep. 8, 284–296 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Ping, X. L. et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 24, 177–189 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Liu, J. et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).

    CAS  PubMed  Google Scholar 

  11. 11.

    Patil, D. P. et al. m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537, 369–373 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Jia, G. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Zheng, G. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18–29 (2013).

    CAS  PubMed  Google Scholar 

  14. 14.

    Mauer, J. et al. Reversible methylation of m6Am in the 5′ cap controls mRNA stability. Nature 541, 371–375 (2017).

    CAS  PubMed  Google Scholar 

  15. 15.

    Liu, N. et al. N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518, 560–564 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Zhou, K. I. et al. N6-methyladenosine modification in a long noncoding RNA hairpin predisposes Its conformation to protein binding. J. Mol. Biol. 428, 822–833 (2016).

    CAS  PubMed  Google Scholar 

  17. 17.

    Konig, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Alarcon, C. R. et al. HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events. Cell 162, 1299–1308 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Wang, X. et al. N6-methyladenosine modulates messenger RNA translation efficiency. Cell 161, 1388–1399 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Wang, X. et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117–120 (2014).

    PubMed  Google Scholar 

  21. 21.

    Du, H. et al. YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex. Nat. Commun. 7, 12626 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Kennedy, E. M. et al. Posttranscriptional m6A Editing of HIV-1 mRNAs Enhances Viral Gene Expression. Cell Host Microbe 19, 675–685 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Shi, H. et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA. Cell Res. 27, 315–328 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Li, A. et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation. Cell Res. 27, 444–447 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Xiao, W. et al. Nuclear m6A reader YTHDC1 regulates mRNA splicing. Mol. Cell 61, 507–519 (2016).

    CAS  PubMed  Google Scholar 

  26. 26.

    Hsu, P. J. et al. Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis. Cell Res. 27, 1115–1127 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Lichinchi, G. et al. Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells. Nat. Microbiol. 1, 16011 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Gokhale, N. S. et al. N6-Methyladenosine in Flaviviridae Viral RNA genomes regulates infection. Cell Host Microbe 20, 654–665 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Lichinchi, G. et al. Dynamics of human and viral RNA methylation during Zika virus infection. Cell Host Microbe 20, 666–673 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Tirumuru, N. et al. N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. eLife 5, e15528 (2016).

  31. 31.

    Ye, F., Chen, E. R. & Nilsen, T. W. Kaposi’s sarcoma-associated herpesvirus utilizes and manipulates RNA N6-adenosine methylation to promote lytic replication. J. Virol. 91, e00466-17 (2017).

  32. 32.

    Zhou, F. C. et al. Efficient infection by a recombinant Kaposi’s sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis. J. Virol. 76, 6185–6196 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Meng, J., Cui, X., Rao, M. K., Chen, Y. & Huang, Y. Exome-based analysis for RNA epigenome sequencing data. Bioinformatics 29, 1565–1567 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Brulois, K. F. et al. Construction and manipulation of a new Kaposi’s sarcoma-associated herpesvirus bacterial artificial chromosome clone. J. Virol. 86, 9708–9720 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Nakamura, H. et al. Global changes in Kaposi’s sarcoma-associated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator. J. Virol. 77, 4205–4220 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Jones, T. et al. Direct and efficient cellular transformation of primary rat mesenchymal precursor cells by KSHV. J. Clin. Invest. 122, 1076–1081 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Lee, M. S. et al. Human mesenchymal stem cells of diverse origins support persistent infection with Kaposi’s sarcoma-associated herpesvirus and manifest distinct angiogenic, invasive, and transforming phenotypes. mBio 7, e02109–15 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Xie, J., Ajibade, A. O., Ye, F., Kuhne, K. & Gao, S. J. Reactivation of Kaposi’s sarcoma-associated herpesvirus from latency requires MEK/ERK, JNK and p38 multiple mitogen-activated protein kinase pathways. Virology 371, 139–154 (2008).

    CAS  PubMed  Google Scholar 

  39. 39.

    Greene, W. & Gao, S. J. Actin dynamics regulate multiple endosomal steps during Kaposi’s sarcoma-associated herpesvirus entry and trafficking in endothelial cells. PLoS Pathog. 5, e1000512 (2009).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Davis, D. A. et al. Hypoxia induces lytic replication of Kaposi’s sarcoma-associated herpesvirus. Blood 97, 3244–3250 (2001).

    CAS  PubMed  Google Scholar 

  41. 41.

    Hollingworth, R. et al. Activation of DNA damage response pathways during lytic replication of KSHV. Viruses 7, 2908–2927 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Meyer, K. D. & Jaffrey, S. R. Rethinking m6A readers, writers, and erasers. Annu. Rev. Cell Dev. Biol. 33, 319–342 (2017).

  43. 43.

    Moss, B., Gershowitz, A., Stringer, J. R., Holland, L. E. & Wagner, E. K. 5′-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA. J. Virol. 23, 234–239 (1977).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Covarrubias, S. et al. Coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease Xrn1. PLoS Pathog. 7, e1002339 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Chandriani, S. & Ganem, D. Host transcript accumulation during lytic KSHV infection reveals several classes of host responses. PLoS ONE 2, e811 (2007).

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Chen, T., You, Y., Jiang, H. & Wang, Z. Z. Epithelial-mesenchymal transition (EMT): a biological process in the development, stem cell differentiation, and tumorigenesis. J. Cell Physiol. 232, 3261–3272 (2017).

  47. 47.

    Moustakas, A. & Heldin, C. H. Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression. Cancer Sci. 98, 1512–1520 (2007).

    CAS  PubMed  Google Scholar 

  48. 48.

    Meyer, K. D. et al. 5′ UTR m6A promotes cap-independent translation. Cell 163, 999–1010 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Moody, R. et al. KSHV microRNAs mediate cellular transformation and tumorigenesis by redundantly targeting cell growth and survival pathways. PLoS Pathog. 9, e1003857 (2013).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Brulois, K. & Jung, J. U. Interplay between Kaposi’s sarcoma-associated herpesvirus and the innate immune system. Cytokine Growth Factor Rev. 25, 597–609 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Lee, M. S. et al. Exploitation of the complement system by oncogenic Kaposi’s sarcoma-associated herpesvirus for cell survival and persistent infection. PLoS Pathog. 10, e1004412 (2014).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Cheng, F. et al. Screening of the human kinome identifies MSK1/2-CREB1 as an essential pathway mediating Kaposi’s sarcoma-associated herpesvirus lytic replication during primary infection. J. Virol. 89, 9262–9280 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Gao, S. J., Deng, J. H. & Zhou, F. C. Productive lytic replication of a recombinant Kaposi’s sarcoma-associated herpesvirus in efficient primary infection of primary human endothelial cells. J. Virol. 77, 9738–9749 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Dominissini, D., Moshitch-Moshkovitz, S., Salmon-Divon, M., Amariglio, N. & Rechavi, G. Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing. Nat. Protoc. 8, 176–189 (2013).

  55. 55.

    Arias, C. et al. KSHV 2.0: a comprehensive annotation of the Kaposi’s sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features. PLoS Pathog. 10, e1003847 (2014).

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Bai, Z. et al. Genomewide mapping and screening of Kaposi’s sarcoma-associated herpesvirus (KSHV) 3′ untranslated regions identify bicistronic and polycistronic viral transcripts as frequent targets of KSHV microRNAs. J. Virol. 88, 377–392 (2014).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Majerciak, V. et al. A viral genome landscape of RNA polyadenylation from KSHV latent to lytic infection. PLoS Pathog. 9, e1003749 (2013).

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Pearce, M., Matsumura, S. & Wilson, A. C. Transcripts encoding K12, v-FLIP, v-cyclin, and the microRNA cluster of Kaposi’s sarcoma-associated herpesvirus originate from a common promoter. J. Virol. 79, 14457–14464 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Tang, S. & Zheng, Z. M. Kaposi’s sarcoma-associated herpesvirus K8 exon 3 contains three 5′-splice sites and harbors a K8.1 transcription start site. J. Biol. Chem. 277, 14547–14556 (2002).

    CAS  PubMed  Google Scholar 

  60. 60.

    Yamanegi, K., Tang, S. & Zheng, Z. M. Kaposi’s sarcoma-associated herpesvirus K8beta is derived from a spliced intermediate of K8 pre-mRNA and antagonizes K8alpha (K-bZIP) to induce p21 and p53 and blocks K8alpha-CDK2 interaction. J. Virol. 79, 14207–14221 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61.

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    PubMed  PubMed Central  Google Scholar 

  62. 62.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Meth. 9, 357–359 (2012).

    CAS  Google Scholar 

  63. 63.

    Meng, J. et al. A protocol for RNA methylation differential analysis with MeRIP-Seq data and exomePeak R/Bioconductor package. Methods 69, 274–281 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Krishnamoorthy, K. & Thomson, J. A more powerful test for comparing two Poisson means. J. Stat. Plan. Inf. 119, 23–35 (2004).

    Google Scholar 

  65. 65.

    Cui, X. et al. Guitar: An R/Bioconductor package for gene annotation guided transcriptomic analysis of RNA-related genomic features. Biomed. Res. Int. 2016, 8367534 (2016).

    PubMed  PubMed Central  Google Scholar 

  66. 66.

    Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67.

    Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat. Biotechnol. 31, 46–53 (2013).

    CAS  PubMed  Google Scholar 

  68. 68.

    Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank S.-J.G.’s laboratory for technical assistance and helpful discussions. This work was supported by grants from the NIH (CA096512, CA124332, CA132637, CA213275, CA177377, DE025465 and CA197153) to S.-J.G., and (GM113245) to Y.H.

Author information

Affiliations

Authors

Contributions

Conceptualization, B.T., H.L., Y.H., S.-J.G.; investigation, B.T., S.R.D.S., O.S., H.Y.; methodology, B.T., H.L., S.Z., Y.H., S.-J.G.; formal analysis, H.L., S.Z., L.Z., J.M., X.C., S.-W.Z., Y.H.; writing—original draft, B.T., H.L., S.-J.G.; writing—review and editing, B.T., H.L., S.Z., S.R.D.S., L.Z., J.M., X.C., O.S., H.Y., S.-W.Z., Y.H., S.-J.G.; supervision and management, Y.H., S.-J.G.; funding acquisition, Y.H., S.-J.G.

Corresponding authors

Correspondence to Yufei Huang or Shou-Jiang Gao.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

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

Electronic supplementary material

Supplementary Information

Supplementary Figures 1–10, Supplementary Tables 3 and 8.

Life Sciences Reporting Summary

Supplementary Table 1

Latent KSHV m6A/m peaks in five types of cells latently infected by KSHV.

Supplementary Table 2

Latent and lytic KSHV m6A/m peaks in KiSLK and BCBL1-R cells.

Supplementary Table 4

Differential cellular methylation and gene expression in uninfected cells and cells latently infected by KSHV.

Supplementary Table 5

Significantly enriched pathways of hypomethylated and hypermethylated cellular genes following latent KSHV infection.

Supplementary Table 6

Differential cellular methylation and gene expression in KSHV-infected cells induced for lytic replication for 48 h compared to uninduced cells in KiSLK and BCBL1-R cells.

Supplementary Table 7

Significantly enriched pathways of 5′ UTR hypomethylated and 3′ UTR hypermethylated cellular genes in KSHV-infected cells induced for lytic replication for 48 h compared to uninduced cells in KiSLK and BCBL1-R cells.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tan, B., Liu, H., Zhang, S. et al. Viral and cellular N6-methyladenosine and N6,2′-O-dimethyladenosine epitranscriptomes in the KSHV life cycle. Nat Microbiol 3, 108–120 (2018). https://doi.org/10.1038/s41564-017-0056-8

Download citation

Further reading