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PLoS Pathog. 2017 March; 13(3): e1006188.
Published online 2017 March 9. doi:  10.1371/journal.ppat.1006188
PMCID: PMC5344520

RNA modifications go viral

Matthew J Evans, Editor

Introduction

Viral life cycles are often coordinated by precise mechanisms that act on their RNA. For example, the microRNA miR-122 interacts with the viral RNA genome of hepatitis C virus (HCV) and is required for HCV replication [1]. In the past year, several groups have reported a new RNA regulatory control to viral infection—the posttranscriptional RNA modification N6-methyladenosine (m6A). This reversible RNA modification is the most prevalent internal modification of the more than 60 known chemical modifications in eukaryotic RNA. The deposition of m6A on RNA is controlled by cellular m6A machinery comprising methyltransferase and demethylase enzymes, as well as m6A-specific binding proteins (recently reviewed in [2]; Fig 1). By affecting mRNA and noncoding RNA structure, localization, and function, m6A plays an important role in many fundamental biological processes [2].

Fig 1
The cellular m6A machinery.

A role for m6A in viral infection has been hypothesized since the 1970s, when m6A was found on RNA of several viruses [37]. Recently, advances in sequencing-based strategies used to profile m6A have expanded the known repertoire of viruses with m6A in their RNA to include human immunodeficiency virus 1 (HIV-1) and RNA viruses in the family Flaviviridae, such as HCV and Zika virus (ZIKV; Table 1) [812]. In this article, we will review the emerging role for m6A in regulating viral infection.

Table 1
List of viruses known to contain m6A in their RNA.

A historical perspective on m6A in viral RNA

Early work on RNA modifications in the 1970s often used viral systems to characterize RNA modifications, including the mRNA “cap” structures. Such early studies, which used chromatographic analysis of radiolabeled and enzymatically digested RNA, uncovered a high degree of internal m6A modification in cellular mRNA and viral RNAs from the DNA viruses simian virus 40 (SV40), adenovirus-2, and herpes simplex virus 1 [35]. m6A was also found in the viral genomic RNA of multiple retroviruses and in the mRNA from influenza A virus, a negative-stranded RNA virus [6, 7, 1316]. Interestingly, all these viruses have a nuclear stage in their life cycle, which led the field to believe that the nucleus was the primary site of m6A modification of RNA.

Subsequent experiments mapped the m6A sites in viral RNAs, revealing an interesting heterogeneity in viral m6A patterns. Rous sarcoma virus genomic RNA contained 10–15 m6A modifications per molecule, all localized to the 3’ half of genomic RNA, while m6A in SV40 and adenovirus-2 mRNA was near spliced regions [4, 14, 17]. Furthermore, the number of m6A residues on individual segments of influenza A virus mRNAs varied greatly between segments [18]. These viral m6A-mapping studies revealed a putative consensus motif for m6A: GAmC and AAmC, which was also later confirmed in cellular mRNA [17, 19, 20]. Indeed mutation of GAC to GAU in a cluster of two such motifs in Rous sarcoma virus prevented m6A modification at these sites [21, 22]. Modern sequencing techniques to detect m6A based on enrichment of m6A-modified RNA fragments using an m6A-specific antibody (m6A-seq), as well as biochemical analyses of the specificity of the m6A methyltransferase complex, have validated the early findings on viral RNA. The consensus motif for m6A is now known to be DRAmCH (where D = G/A/U, R = G > A, and H = U/C/A) [2325].

Early research on m6A in viral infection pointed to the modification regulating viral RNA splicing. The m6A-methylation inhibitor cycloleucine reduced splicing of the Rous sarcoma virus Env mRNA and impaired the proper nuclear processing and export of SV40 late mRNA [2628]. Furthermore, m6A was proposed to regulate the splicing of adenovirus-2 late transcripts [29]. Indeed, m6A has now been to shown to regulate mRNA splicing, highlighting the value of these early viral studies in uncovering m6A function [30].

Recent advances in m6A in viral RNA

The recent identification of the cellular m6A machinery (see Fig 1) now allows for mechanistic studies on the function of this RNA modification during viral infection [2]. Recently, three groups have found a proviral role for m6A in HIV-1 infection [810]. Interestingly, these studies found that the function of individual m6A sites in HIV-1 RNA can be varied, ranging from regulating HIV-1 RNA nuclear export to enhancing viral gene expression [8, 9]. Furthermore, the m6A-binding cytosolic YTH domain family (YTHDF) proteins were found to bind to HIV-1 RNA at m6A sites [9, 10] but have varied roles in regulating HIV-1 infection, from promoting viral transcript abundance and translation to suppressing viral reverse transcription [9, 10]. Given that m6A regulates splicing during infection by other retroviruses, HIV-1 mRNA splicing may also be affected by m6A and by YTHDC1, a nuclear YTH domain containing m6A-binding protein involved in cellular mRNA splicing [30]. While this work has uncovered m6A and the m6A machinery as important regulators of HIV-1 infection, one general limitation of experiments involving the knockdown of the cellular m6A machinery is that such depletion could affect the expression of pro- or anti-viral host factors, leading to an indirect effect on viral infection. Experiments involving viruses that contain m6A-abrogating mutations will be invaluable in pinpointing the direct role of this modification on viral RNA during infection.

We, and others, have recently found a role for m6A in regulating RNA viruses of the Flaviviridae family. Using m6A-seq, we mapped several regions modified by m6A across the RNA genomes of the Flaviviridae members HCV, ZIKV, dengue virus, yellow fever virus, and West Nile virus [11]. Concurrently, another group also identified m6A in ZIKV RNA [12]. These viral RNAs are the first examples of exclusively cytoplasmic RNA species that contain m6A, indicating that the cellular m6A methyltransferases may be active in the cytoplasm under some cases. Indeed, the m6A methyltransferases are present in the cytoplasm as well as the nucleus [11]. Perhaps they are targeted to viral RNAs by cellular factors yet to be defined that modulate the specificity and localization of the methyltransferase complex. We also tested if m6A had any role in Flaviviridae infection, and found that m6A suppressed the packaging of HCV RNA into infectious viral particles. A conserved cluster of four m6A sites in the HCV E1 gene was the primary driver of this phenotype, such that abrogation of m6A in this region by mutation altered RNA–protein interactions required for viral assembly [11]. Similar to our work, Lichinchi et al. found that m6A also limited ZIKV infection, suggesting that m6A negatively regulates Flaviviridae infection [11, 12]. Both of these studies mapped the Flaviviridae m6A sites at a single time point of infection, catching only a snapshot of the overall m6A profile on the viral genomes. As current m6A-mapping technologies do not allow us to easily determine the m6A occupancy of any individual site or whether it occurs on the same viral RNA species, it is likely that these viral genomes will have divergent m6A sites and occupancies at different stages of their life cycles. For example, we found that virion-associated RNA had less overall m6A than intracellular replicating HCV RNA [11]. By expanding these studies to capture the viral m6A sites over a time course of infection or on specific viral RNA species, we could identify new controls governed by m6A that regulate specific aspects of viral replication, including viral RNA stability, translation, replication, packaging, or even immune evasion (see below). Furthermore, as m6A destabilizes RNA secondary structure [2], it could directly alter cis-regulatory structural elements in RNA virus genomes.

The presence of RNA modifications on viral RNAs may prevent detection by host pattern recognition receptors that trigger antiviral innate immunity. Indeed, two studies have shown that internal m6A modification of in vitro synthesized RNAs ameliorates innate immune activation by the known RNA-sensing pattern recognition receptors TLR3 and RIG-I [31, 32]. Therefore, m6A-modification of the pathogen-associated molecular patterns within viral RNA may be an evolutionary adaptation for immune evasion. Identifying m6A modification in viral RNA pathogen-associated molecular patterns during infection will be critical in proving that m6A serves as a shield on viral RNA to prevent induction of antiviral signaling pathways.

Epitranscriptomic changes to host mRNA during viral infection

Viral infection induces broad changes in the host transcriptome and proteome. Therefore, it is not surprising that viral infection can also alter the m6A-epitranscriptome in host mRNA. Indeed, both HIV-1 and ZIKV impact the host m6A-epitranscriptome with changes to the specific transcripts containing m6A and to the overall m6A-topology [8, 12]. Specifically, during viral infection, the level of m6A increases at the 5′UTR of mRNAs, with a concomitant decrease in m6A modification at 3’UTRs. A similar increase in m6A at 5’UTRs has been reported in heat shock–related transcripts during heat shock, which promotes the translation of these mRNAs [33]. Interestingly, during viral infection, many m6A-altered transcripts are related to viral replication and immune responses [8, 12]. Therefore, m6A modification to specific mRNAs could be virally induced to promote infection, or by the host to restrict infection, allowing for an additional layer of gene expression regulation. Future studies on viral- or host-mediated epitranscriptomic changes and identification of the factors that regulate these altered epitranscriptomes will be essential to understanding how viral infection alters host gene expression.

Conclusions and future perspectives

As important posttranscriptional modulators of RNA function, m6A and other RNA modifications likely regulate infection by all classes of viruses. Recent scientific and technological advances have now set the stage for the systematic exploration of many outstanding questions regarding the role of m6A during viral infection. Going forward, perturbing the host m6A machinery and mutating m6A motifs in viral RNAs will be invaluable techniques used to study the function of m6A on viral RNA structure, localization, splicing, stability, translation, and immune evasion. Furthermore, understanding viral- or host-induced changes in the cellular m6A epitranscriptome will be crucial in understanding gene regulation during viral infection. Indeed, as for many fundamental biological systems, viral infection may prove to be a useful model for understanding how m6A affects cellular RNA expression and function. Therefore, we expect that virology and its exciting discoveries will be at the heart of the renaissance of m6A and RNA modification research.

Acknowledgments

We apologize to those whose seminal work we are not able to cite due to space limitations. We thank the members of the Horner Lab for a critical reading of this manuscript.

Funding Statement

Research in the Horner Lab is supported by the National Institutes of Health (R01AI125416, R21NS100545, and R21AI124100), the Duke University Center for AIDS Research (P30AI064518), and a Duke School of Medicine Whitehead Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1. Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science. 2005;309(5740):1577–81. doi: 10.1126/science.1113329 [PubMed]
2. Zhao BS, Roundtree IA, He C. Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol. 2017;18(1):31–42. doi: 10.1038/nrm.2016.132 [PMC free article] [PubMed]
3. Lavi S, Shatkin AJ. Methylated simian virus 40-specific RNA from nuclei and cytoplasm of infected BSC-1 cells. Proc Natl Acad Sci U S A. 1975;72(6):2012–6. [PubMed]
4. Sommer S, Salditt-Georgieff M, Bachenheimer S, Darnell JE, Furuichi Y, Morgan M, et al. The methylation of adenovirus-specific nuclear and cytoplasmic RNA. Nucleic Acids Res. 1976;3(3):749–65. [PMC free article] [PubMed]
5. Moss B, Gershowitz A, Stringer JR, Holland LE, Wagner EK. 5'-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA. J Virol. 1977;23(2):234–9. [PMC free article] [PubMed]
6. Furuichi Y, Shatkin AJ, Stavnezer E, Bishop JM. Blocked, methylated 5'-terminal sequence in avian sarcoma virus RNA. Nature. 1975;257(5527):618–20. [PubMed]
7. Krug RM, Morgan MA, Shatkin AJ. Influenza viral mRNA contains internal N6-methyladenosine and 5'-terminal 7-methylguanosine in cap structures. J Virol. 1976;20(1):45–53. [PMC free article] [PubMed]
8. Lichinchi G, Gao S, Saletore Y, Gonzalez GM, Bansal V, Wang Y, et al. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells. Nature microbiology. 2016;1(4):16011. [PubMed]
9. Kennedy EM, Bogerd HP, Kornepati AV, Kang D, Ghoshal D, Marshall JB, et al. Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression. Cell Host Microbe. 2016;19(5):675–85. doi: 10.1016/j.chom.2016.04.002 [PMC free article] [PubMed]
10. Tirumuru N, Zhao BS, Lu W, Lu Z, He C, Wu L. N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. Elife. 2016;5. [PMC free article] [PubMed]
11. Gokhale NS, McIntyre AB, McFadden MJ, Roder AE, Kennedy EM, Gandara JA, et al. N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection. Cell Host Microbe. 2016;20(5):654–65. doi: 10.1016/j.chom.2016.09.015 [PMC free article] [PubMed]
12. Lichinchi G, Zhao BS, Wu Y, Lu Z, Qin Y, He C, et al. Dynamics of Human and Viral RNA Methylation during Zika Virus Infection. Cell Host Microbe. 2016;20(5):666–73. doi: 10.1016/j.chom.2016.10.002 [PubMed]
13. Stoltzfus CM, Dimock K. Evidence of methylation of B77 avian sarcoma virus genome RNA subunits. J Virol. 1976;18(2):586–95. [PMC free article] [PubMed]
14. Beemon K, Keith J. Localization of N6-methyladenosine in the Rous sarcoma virus genome. J Mol Biol. 1977;113(1):165–79. [PubMed]
15. Thomason AR, Brian DA, Velicer LF, Rottman FM. Methylation of high-molecular-weight subunit RNA of feline leukemia virus. J Virol. 1976;20(1):123–32. [PMC free article] [PubMed]
16. Bondurant M, Hashimoto S, Green M. Methylation pattern of genomic RNA from Moloney murine leukemia virus. J Virol. 1976;19(3):998–1005. [PMC free article] [PubMed]
17. Canaani D, Kahana C, Lavi S, Groner Y. Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA. Nucleic Acids Res. 1979;6(8):2879–99. [PMC free article] [PubMed]
18. Narayan P, Ayers DF, Rottman FM, Maroney PA, Nilsen TW. Unequal distribution of N6-methyladenosine in influenza virus mRNAs. Mol Cell Biol. 1987;7(4):1572–5. [PMC free article] [PubMed]
19. Dimock K, Stoltzfus CM. Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits. Biochemistry. 1977;16(3):471–8. [PubMed]
20. Wei CM, Gershowitz A, Moss B. 5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA. Biochemistry. 1976;15(2):397–401. [PubMed]
21. Kane SE, Beemon K. Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing. Mol Cell Biol. 1985;5(9):2298–306. [PMC free article] [PubMed]
22. Kane SE, Beemon K. Inhibition of methylation at two internal N6-methyladenosine sites caused by GAC to GAU mutations. J Biol Chem. 1987;262(7):3422–7. [PubMed]
23. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 2012;149(7):1635–46. doi: 10.1016/j.cell.2012.05.003 [PMC free article] [PubMed]
24. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485(7397):201–6. doi: 10.1038/nature11112 [PubMed]
25. Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10(2):93–5. doi: 10.1038/nchembio.1432 [PMC free article] [PubMed]
26. Dimock K, Stolzfus CM. Cycloleucine blocks 5'-terminal and internal methylations of avian sarcoma virus genome RNA. Biochemistry. 1978;17(17):3627–32. [PubMed]
27. Stoltzfus CM, Dane RW. Accumulation of spliced avian retrovirus mRNA is inhibited in S-adenosylmethionine-depleted chicken embryo fibroblasts. J Virol. 1982;42(3):918–31. [PMC free article] [PubMed]
28. Finkel D, Groner Y. Methylations of adenosine residues (m6A) in pre-mRNA are important for formation of late simian virus 40 mRNAs. Virology. 1983;131(2):409–25. [PubMed]
29. Chen-Kiang S, Nevins JR, Darnell JE Jr. N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA. J Mol Biol. 1979;135(3):733–52. [PubMed]
30. Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, et al. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol Cell. 2016;61(4):507–19. doi: 10.1016/j.molcel.2016.01.012 [PubMed]
31. Kariko K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005;23(2):165–75. doi: 10.1016/j.immuni.2005.06.008 [PubMed]
32. Durbin AF, Wang C, Marcotrigiano J, Gehrke L. RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling. mBio. 2016;7(5). [PMC free article] [PubMed]
33. Zhou J, Wan J, Gao X, Zhang X, Jaffrey SR, Qian SB. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature. 2015;526(7574):591–4. doi: 10.1038/nature15377 [PMC free article] [PubMed]
34. Mauer J, Luo X, Blanjoie A, Jiao X, Grozhik AV, Patil DP, et al. Reversible methylation of m6Am in the 5' cap controls mRNA stability. Nature. 2016. [PubMed]

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