RNA modifications go viral.

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.

N6-methyladenosine (m6A) is a reversible RNA modification that occurs in cellular and viral RNA. The deposition of m6A at the consensus motif DRAmCH (where D = G/A/U, R = G > A, and H = U/C/A) is governed by a cellular methyltransferase complex composed of the “writers” METTL3 and METTL14, and other noncatalytic cofactors. m6A modification can be reversed by the “erasers” FTO and ALKBH5. *We note that FTO has recently been found to have greater specificity for the m6Am modifications present in mRNA cap structures than for m6A [34]. “Reader” m6A-specific RNA binding proteins, including the cytoplasmic YTHDF1, YTHDF2, YTHDF3, and nuclear YTHDC1 control the function of m6A on RNA. YTHDF1 promotes translation of cellular m6A-mRNAs, while YTHDF2 targets them for degradation. YTHDC1 regulates the splicing of m6A-modified pre-mRNA. The role of m6A and the m6A machinery in RNA function and biological processes is further reviewed in [2].

A role for m6A in viral infection has been hypothesized since the 1970s, when m6A was found on RNA of several viruses [3-7]. 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) [8-12]. 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.

Virus	Summary of knowledge	References
DNA viruses
Simian virus 40	Viral late transcripts have ~3 internal m6A residues. Blocking m6A with cycloleucine impairs nuclear processing and export of late viral mRNAs.	[3, 17, 28]
Adenovirus-2	Viral RNAs contain internal m6A. Prior to splicing, viral RNA is modified by m6A. m6A is retained in the viral mRNA after nuclear export.	[4, 29]
Herpes simplex virus	Viral mRNAs contain internal m6A.	[5]
Retroviruses
HIV-1	Viral mRNA and genomes contain m6A, which is concentrated at 3’ regions. m6A sites at the Rev-response element RNA structure alter nuclear export of viral RNA. m6A-binding YTHDF proteins bind to viral RNA, promote viral replication, and may suppress genomic RNA reverse transcription following infection.	[8, 9, 10]
Rous sarcoma virus	Genomic RNA has ~10–15 m6A residues per molecule mostly in the 3’ terminal third of the genomic RNA. Blocking m6A by cycloleucine reduces formation of the mature, spliced Env mRNA.	[6, 13, 14, 19, 21, 22, 26, 27]
Feline leukemia virus	Genomic RNA contains internal m6A modification.	[15]
Moloney murine leukemia virus	Genomic RNA contains internal m6A modification.	[16]
(+)-stranded RNA virus
HCV	The viral RNA genome has multiple internal m6A sites. m6A suppresses viral particle production, but does not affect viral RNA replication. YTHDF proteins suppress viral particle production, and relocalize to viral assembly sites around lipid droplets. Mutation of one cluster of m6A sites increases viral particle production.	[11]
ZIKV	The viral RNA genome has multiple internal m6A sites, with differences in m6A modification patterns in 3 strains. m6A and YTHDF proteins suppress viral infection.	[11, 12]
Dengue, Yellow fever, and West Nile virus	The viral RNA genome has multiple internal m6A sites.	[11]
(-)-stranded RNA viruses
Influenza A virus	Viral mRNAs and genomic RNA segments have internal m6A. m6A is unequally distributed on viral mRNAs.	[7, 18]

YTHDF, YTH domain family.

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 [3-5]. 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, 13–16]. 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) [23-25].

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 [26-28]. 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 [8-10]. 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.