m5C Goes Viral
Authors: Shelby Winans, Karen Beemon
Journal: Cell Host & Microbe (2019)

In this issue of Cell Host & Microbe, Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar find that HIV-1 genomic RNA has much more m5C than cellular mRNA. Deleting the m5C "writer" NSUN2 decreases HIV-1 m5C levels, promotes translation of the HIV-1 5′ gag gene, and alters splicing at the A2 site. In this issue of Cell Host & Microbe, Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar find that HIV-1 genomic RNA has much more m5C than cellular mRNA. Deleting the m5C "writer" NSUN2 decreases HIV-1 m5C levels, promotes translation of the HIV-1 5′ gag gene, and alters splicing at the A2 site. The field of epitranscriptomics has burgeoned over the past decade with the advent of new, high-throughput technologies to detect RNA modifications. While work on modifications of RNA dates back over 60 years, most early work focused on the heavily modified and abundant ribosomal and tRNA transcripts. mRNA was found to have a 5′ cap (with m7G and 2'0-methylated bases) and internal N6-methyladenosine (m6A) in 1975. Initial studies identified m6A in many mRNA substrates, but the biological significance of such modifications was not known. The advent of methylated RNA immunoprecipitation and sequencing (MeRIP-seq) techniques in 2012 brought renewed interest in study of m6A on a global scale. In addition, m6A "writers, readers, and erasers" have been identified, and m6A has been shown to have significant impact on cellular mRNA stability, splicing, and translation (Shi et al., 2019Shi H. Wei J. He C. Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers.Mol. Cell. 2019; 74: 640-650Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). m6A has also been shown to have an impact on viral replication. m6A residues have been identified in the genomic RNA or mRNA transcripts of many viruses, including retroviruses, DNA viruses, influenza, and even RNA viruses that replicate in the cytoplasm (reviewed in Williams et al., 2019Williams G.D. Gokhale N.S. Horner S.M. Regulation of viral infection by the RNA modification N6-methyladenosine.Annu Rev Virol. 2019; https://doi.org/10.1146/annurev-virology-092818-015559Crossref PubMed Scopus (70) Google Scholar). Recently, several labs have mapped m6A in HIV-1 RNA and observed differences in its localization as well as varying functions. While m6A has been shown to promote Rev-mediated HIV-1 RNA export from the nucleus and to stabilize HIV-1 RNA, it has also been seen to inhibit reverse transcription (Williams et al., 2019Williams G.D. Gokhale N.S. Horner S.M. Regulation of viral infection by the RNA modification N6-methyladenosine.Annu Rev Virol. 2019; https://doi.org/10.1146/annurev-virology-092818-015559Crossref PubMed Scopus (70) Google Scholar). In this issue of Cell Host & Microbe, Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar examine nine different epitranscriptomic modifications of HIV-1 genomic RNA (gRNA), using a quantitative ultrahigh-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) technique. Levels of modification of HIV-1 RNA are compared to total cellular mRNA analyzed in parallel. They use highly purified, packaged HIV-1 genomic RNA for this analysis. m6A is the most abundant internal mRNA modification in cellular mRNAs, and levels in HIV-1 virion RNA are similar but about 40% higher than average cellular mRNA, when adjusted for size and base composition. HIV-1 genomic RNA was found to have an average of 13 m6A residues, similar to that seen for Rous sarcoma virus (Beemon and Keith, 1977Beemon K. Keith J. Localization of N6-methyladenosine in the Rous sarcoma virus genome.J. Mol. Biol. 1977; 113: 165-179Crossref PubMed Scopus (127) Google Scholar). In contrast, murine leukemia virus (MLV) genomic RNA analyzed similarly had 51 m6A residues (Courtney et al., 2019bCourtney D.G. Chalem A. Bogerd H.P. Law B.A. Kennedy E.M. Holley C.L. Cullen B.R. Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression.MBio. 2019; 10https://doi.org/10.1128/mBio.01209-19Crossref PubMed Scopus (32) Google Scholar). Surprisingly, the level of m5C and 2'0-methylations in HIV-1 RNA are over 10× higher than those in average cellular mRNAs (Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). While m5C is quite rare in cell mRNAs (.05% of Cs), it varies from 0.6% to 1.4% of Cs in HIV-1 RNA. This represents an average of 11 m5C per HIV-1 genomic RNA. In addition, 2'O-methylated bases each represent about 1% of the cognate base in HIV-1 RNA. Similarly high levels of m5C and 2'O-methylations are seen for MLV RNA (Courtney et al., 2019bCourtney D.G. Chalem A. Bogerd H.P. Law B.A. Kennedy E.M. Holley C.L. Cullen B.R. Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression.MBio. 2019; 10https://doi.org/10.1128/mBio.01209-19Crossref PubMed Scopus (32) Google Scholar). Importantly, 2'O-methylation of HIV-1 RNA has recently been shown to aid retroviral replication by allowing evasion of innate immune recognition (Ringeard et al., 2019Ringeard M. Marchand V. Decroly E. Motorin Y. Bennasser Y. FTSJ3 is an RNA 2′-O-methyltransferase recruited by HIV to avoid innate immune sensing.Nature. 2019; 565: 500-504Crossref PubMed Scopus (101) Google Scholar). Studies of m5C function in cell mRNAs have shown that it can play a role in regulating nuclear export. Depletion of a proposed m5C reader protein, ALYREF, resulted in nuclear retention of m5C-modified mRNAs without affecting non-modified transcripts (Yang et al., 2017Yang X. Yang Y. Sun B.F. Chen Y.S. Xu J.W. Lai W.Y. Li A. Wang X. Bhattarai D.P. Xiao W. et al.5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.Cell Res. 2017; 27: 606-625Crossref PubMed Scopus (405) Google Scholar). While m5C has also been proposed to regulate translation, the specific effects of the modification are variable and likely depend on the context of the modification (reviewed in Trixl and Lusser, 2019Trixl L. Lusser A. The dynamic RNA modification 5-methylcytosine and its emerging role as an epitranscriptomic mark.Wiley Interdiscip. Rev. RNA. 2019; 10: e1510Crossref PubMed Scopus (146) Google Scholar). Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar utilize an antibody-based photoactivated crosslinking method (PA-m5C-seq) to map the locations of the m5C residues on the viral genomic RNA. 18 distinct and reproducible m5C peaks are discovered, indicating that the genomic RNA is heterogeneously methylated. Further, the m5C modified residues are more prevalent at the 3′ end of the viral RNA, a surprising finding in light of previous studies that have identified m5C enrichment near translation start sites in cellular mRNAs (Yang et al., 2017Yang X. Yang Y. Sun B.F. Chen Y.S. Xu J.W. Lai W.Y. Li A. Wang X. Bhattarai D.P. Xiao W. et al.5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader.Cell Res. 2017; 27: 606-625Crossref PubMed Scopus (405) Google Scholar). m5C residues mapped to the HIV-1 genome appeared similar in location in virions and in the cell (Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Seven members of the NSUN family are known to add methyl groups to RNA. To identify the specific writer protein responsible for methylation, the authors make use of a clever technique in which mutation of an active site residue of the NSUN protein causes the protein to become crosslinked to the RNA that is being modified. By expressing these mutated NSUN proteins and subsequently assaying for NSUN protein presence on packaged viral gRNA, the authors are able to determine that NSUN2 is predominantly responsible for methylation of the HIV-1 genomic RNA. To further validate this, NSUN2 knockout cell lines were infected with HIV-1. In the absence of NSUN2, all but three of the detectable m5C modified residues in the HIV-1 gRNA disappear, further supporting NSUN2 as the predominant cellular factor responsible for m5C modification of HIV-1 RNA. In the absence of NSUN2, viral protein translation is significantly diminished, while viral mRNA levels are comparable in wild-type and NSUN2 knockout cells, indicating that m5C plays a role in post-transcriptional regulation of viral gene expression. They further show that lack of m5C does not affect HIV-1 RNA nuclear export but rather ribosome loading. In the future, it would be interesting to determine whether this is through cap-independent translation initiation. Further, in the absence of NSUN2, alternative splicing of the HIV-1 viral RNA is altered. Splice acceptor site A2, which has an m5C site 150 nucleotides upstream, is utilized less in the absence of NSUN2, thereby implicating m5C in splicing regulation. Why is HIV-1 RNA so heavily modified? The single, full-length retroviral RNA transcript has multiple functions (Leblanc et al., 2013Leblanc J. Weil J. Beemon K. Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA.Wiley Interdiscip. Rev. RNA. 2013; 4: 567-580Crossref PubMed Scopus (37) Google Scholar). It serves as genomic RNA, packaged into viral particles. It is also the major mRNA for Gag and Pol proteins. Third, a fraction of the transcripts are alternatively spliced to generate about 50 different mRNAs. The 5′ end of the viral RNA can form different conformations that favor packaging or translation. Remarkably, addition of a single G residue at the 5′ end of the genome favors a structure that is packaged as dimers rather than translated (Kharytonchyk et al., 2016Kharytonchyk S. Monti S. Smaldino P.J. Van V. Bolden N.C. Brown J.D. Russo E. Swanson C. Shuey A. Telesnitsky A. Summers M.F. Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome.Proc. Natl. Acad. Sci. USA. 2016; 113: 13378-13383Crossref PubMed Scopus (60) Google Scholar). When two or three terminal Gs are transcribed, translation of monomeric RNAs is favored. Thus, it seems possible that epitranscriptomic changes may also be important for this distinction between different fates for the HIV-1 RNA. In fact, Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar report differences in m6A localization between genomic RNA and HIV-1 mRNA in cells. The work of Courtney et al., 2019aCourtney D.G. Tsai K. Bogerd H.P. Kennedy E.M. Law B.A. Emery A. Swanstrom R. Holley C.L. Cullen B.R. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene Expression.Cell Host Microbe. 2019; 26 (this issue): 217-227Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar provides important insight into how the RNA modification m5C regulates retroviral replication. The Cullen lab has also recently shown that m5C regulates the gene expression and replication of another retrovirus, MLV (Courtney et al., 2019bCourtney D.G. Chalem A. Bogerd H.P. Law B.A. Kennedy E.M. Holley C.L. Cullen B.R. Extensive Epitranscriptomic Methylation of A and C Residues on Murine Leukemia Virus Transcripts Enhances Viral Gene Expression.MBio. 2019; 10https://doi.org/10.1128/mBio.01209-19Crossref PubMed Scopus (32) Google Scholar), suggesting that m5C modification may be a general strategy that retroviruses employ to enhance replication. It remains to be seen whether, like m6A, m5C also serves to regulate replication of other viral families. Additionally, it will be important to determine how different modifications interact to regulate viral replication. As the authors argue, selective pressure on viral genomes is so significant that viruses must adapt to have the optimal modifications to maximize their replication potential. By studying the modifications of viral RNAs, we may better understand the functional significance of RNA modifications. Epitranscriptomic Addition of m5C to HIV-1 Transcripts Regulates Viral Gene ExpressionCourtney et al.Cell Host & MicrobeAugust 14, 2019In BriefCourtney et al. report that HIV-1 transcripts are modified by the addition of 5-methylcytosine (m5C) residues. The nuclear methyltransferase NSUN2 is the primary m5C writer and is required for appropriate HIV-1 translation. NSUN2 deficiency and concomitant loss of m5C residues inhibits ribosomal recruitment to and alternative splicing of HIV-1 mRNAs. Full-Text PDF Open Archive