dynamics of the human and viral m 6 a rna methylomes during … · 2017-12-23 · dynamics of the...

Dynamics of the human and viral m6A RNAmethylomes during HIV-1 infection of T cellsGianluigi Lichinchi1,2, Shang Gao1, Yogesh Saletore3, Gwendolyn Michelle Gonzalez4, Vikas Bansal1,Yinsheng Wang4, Christopher E. Mason3,5 and Tariq M. Rana1,2,6*

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of thefunction of m6A in the immune system or its role in host–pathogen interactions. Here, we investigate the topology,dynamics and bidirectional influences of the viral–host RNA methylomes during HIV-1 infection of human CD4 T cells. Weshow that viral infection triggers a massive increase in m6A in both host and viral mRNAs. In HIV-1 mRNA, we identified14 methylation peaks in coding and noncoding regions, splicing junctions and splicing regulatory sequences. We alsoidentified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enrichedfor functions in viral gene expression. The functional relevance of m6A for viral replication was demonstrated by silencingof the m6A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore,methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev response element (RRE) RNA enhancedbinding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a newmechanism for the control of HIV-1 replication and its interaction with the host immune system.

RNA plays numerous vital roles in cellular processes rangingfrom the transfer of genetic information from DNA toprotein to the epigenetic modulation of gene transcription1.

In a similar manner to proteins and DNA, RNA undergoes chemicalmodifications that can influence its metabolism, function and local-ization. More than 100 diverse chemical groups are known tomodify RNA at one or more of its four nucleotides (A, G, C and U).Ribosomal and tRNA sequences incorporate most of the diversechemical modifications. Other than the 5′-cap structure, mRNAsand lncRNAs contain m6A and 5-methyl cytosine (m5C)2,3.Methylation of adenosine at the N6 position (m6A), which wasfirst reported ∼40 years ago4–7, is the most prevalent internal modi-fication of eukaryotic mRNA and contributes to its generation,localization and function8–10. Although our understanding of theimportance of m6A to physiological and pathological processesis increasing rapidly11–13, little is known of the function ofm6A in the mammalian immune system or its role inhost–pathogen interactions.

Adenosine methylation is catalysed by a ‘writer’, which is a large1 MDa RNA methyltransferase complex (MTase) composed of twocatalytic subunits (METTL3 and METTL14), a splicing factor(WTAP), a novel protein (KIAA1429) and other subunits not yetidentified10,14–16. Conversely, methyl groups are removed by two‘erasers’, the RNA demethylases, FTO and AlkBH5 (refs 12,13).The development of techniques to selectively immunoprecipitatem6A and sequence the associated RNA (MeRIP-seq) has beenpivotal to our understanding of the topology and prevalence ofm6A in various eukaryotic transcriptomes8,9. Also, the mRNAregions most highly enriched in m6A are transcription start sites,stop codons and 3′UTRs8–10, raising many hypotheses about their

roles. Methylation of RNA adenosines is a dynamic process andthe exact sites and frequency of modification may vary incells exposed to non-physiological conditions such as stress,inflammation and infection.

Although the precise molecular functions of m6A and itsdynamic regulation are not yet fully understood, it has been postu-lated to play several important roles in RNA metabolism, includingtranslation, stability, splicing, transport and localization17,18. Forexample, the enrichment of m6A in regions of the 3′UTR plays arole in RNA stability through specific recognition of m6A sites byRNA-binding proteins involved in mRNA decay pathways19–21.m6A modification is involved in triggering RNA structural switchesto affect RNA–protein interactions22 as well as directing transla-tional control of heat shock responses23. Additionally, nuclearexport of mRNA is promoted by the increased abundance of m6Aresulting from AlkBH5 depletion13. m6A is enriched in exonicregions flanking splicing sites24. Increased m6A resulting fromFTO knockout favours the binding of splicing factor SRSF2 tomRNA splicing sites and in turn promotes inclusion of targetexons24. Circadian gene expression is delayed upon METTL3depletion25, and the pluripotency of embryonic stem cells is affectedby modulation of METTL3 (refs 21,26) and METTL14 (ref. 27)expression. Taken together, these findings substantiate RNAmethylation as a new epitranscriptomic mechanism for the regu-lation of gene expression.

The internal m6A modification is also present in viral RNA. Thiswas first identified in Rous sarcoma virus28, influenza virus29 andSV40 virus30 several decades ago, but the function and relevanceof this modification to viral replication remain unclear. Similarly,whether and how viral infections alter the dynamics of the host or

1Department of Pediatrics, University of California, San Diego School of Medicine, La Jolla, California 92093, USA. 2Program for RNA Biology and GraduateSchool of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, USA.3Department of Physiology and Biophysics and the Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University, 1305 YorkAvenue, New York, New York 10021, USA. 4Environmental Toxicology Graduate Program and Department of Chemistry, University of California, Riverside,California 92521, USA. 5The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, The Feil Family Brain and MindResearch Institute (BMRI), New York, New York, 10021, USA. 6Institute for Genomic Medicine and Moores Cancer Center, University of CaliforniaSan Diego, La Jolla, California 92093, USA. *e-mail: [email protected]

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viral RNA methylomes remain fundamental and unanswered ques-tions. Notably, m6A has not yet been identified in HIV-1 RNA. Inthis study, we report the presence of this modification in HIV-1RNA and examine the topology, functions and molecular featuresof m6A in both HIV-1 and host RNA during infection of humanCD4+ T cells.

ResultsRNA methylation in T cells is enhanced by HIV-1 infection andmodulates viral replication. To investigate whether HIV-1 RNA ism6A-modified during the course of viral infection, we examined theMT4 human CD4+ T cell line at 3 days post-infection with theHIV-1 LAI clone, the time period when virus is in the activereplication phase. The abundance of m6A was analysed byanti-m6A immunoblotting (Fig. 1a) and quantified by liquidchromatography–tandem mass spectrometry (LC−MS/MS)according to published methods31. Poly(A) RNA from LAI-infected cells showed significantly higher level of m6A (∼30%increase) compared with control uninfected cells (Fig. 1b). Anincrease in methylation was observed in all RNA sizes (Fig. 1a,middle panel), suggesting a widespread effect on cellular mRNAspecies. These results were also confirmed by m6A immunoblotanalysis of poly(A)-enriched RNA where the contribution ofribosomal RNA was removed (Supplementary Fig. 1). Total

RNA from HIV-1-infected cells showed a high molecularweight band with an intense m6A signal, which wassubsequently identified as HIV-1 genomic RNA by northernblot analysis (Fig. 1a, middle and right panels). These resultsdemonstrate that HIV-1 infection of T cells promotesmethylation of both viral and host RNA.

We next determined whether modulation of m6A abundancemight alter the HIV-1 replication rate. MT4 cells were transducedwith shRNAs targeting METTL3, METTL14 or AlkBH5, andknockdown efficiency was assessed by western blot and reversetranscription and quantitative PCR (RT-qPCR) analysis(Supplementary Fig. 2). We chose to deplete AlkBH5 for RNAdemethylase function due to its high expression in T cells.Transduced cells were then infected with HIV-1 LAI and, 3 dayslater, viral replication was monitored by RT-qPCR analysis ofgp120 mRNA levels and p24 immunoblotting of total cell extracts(Fig. 1c,d). Depletion of METTL3 or METTL14 alone significantlydecreased viral replication compared with cells expressing non-targeting control (NTC) shRNA, and an additive effect was observedwhen both MTases were simultaneously knocked down. Conversely,AlkBH5 silencing caused a striking increase in viral replication(Fig. 1c,d). Taken together, these results demonstrate that modu-lation of METTL3, METTL14 and AlkBH5 expression directlyaffects HIV-1 replication.

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Figure 1 | m6A RNA methylation in T cells is promoted by HIV-1 infection and modulates viral replication. a, Analysis of RNA from HIV-1-infected T cells.Total RNA extracted from uninfected (Ctrl) or HIV-1-infected (HIV) MT4 cells 3 days post-infection was analysed by ethidium bromide (EtBr) staining (left),anti-m6A immunoblotting (middle) or northern blotting for HIV-1 RNA (right). Arrow indicates the predicted size of full-length HIV-1 RNA. The data arerepresentative of seven independent experiments. b, LC-MS/MS quantification of m6A content in poly(A)-enriched RNA fractions. Results are the mean ± s.e.m.of five independent experiments. P values were calculated by unpaired two-tailed Student’s t-test. c,d, HIV-1 replication is reduced by METTL3 (M3) andMETTL14 (M14) silencing and enhanced by AlkBH5 silencing. c, RT-qPCR results are shown for gp120 expression in MT4 cells transduced for 3 days withnontargeting control (NTC), METTL3, METTL14 or AlkBH5 shRNA. Cells were then infected with HIV-1 LAI virus and analysed 3 days later. d, Western blotanalysis of HIV-1 p24 intracellular protein expression is shown for the cells described in c. Results are the mean ± s.e.m. of four independent experiments.P values were calculated by unpaired two-tailed Student’s t-test.

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Topology of RNA methylome during HIV-1 infection. We nextexamined the topology of the m6A RNA methylome during HIV-1infection by MeRIP-seq experiments. Poly(A)-enriched RNA fromuninfected and HIV-1-infected MT4 cells was fragmented into60–200 nucleotides and immunoprecipitated using an m6A-specific antibody (see Methods), and the associated RNA was thensequenced. Reads were mapped onto the HIV-1 and humangenomes to identify regions enriched in m6A. We found ∼49% ofinput RNA-seq reads and ∼42% of MeRIP-seq reads mapped tothe HIV genome, respectively (Supplementary Table 1). Weidentified 14 distinct methylation peaks located in coding andnoncoding regions, splicing junctions (D1) and splicing regulatorysequences (Fig. 2a and Table 1), with each peaks suggestingpotential specific functional roles for each methylation peak.Peak 1 encompasses the primary binding site sequence,dimerization sequence, encapsidation sequence (ψ), D1 splicingsite and gag gene start site. Peak 2 is within the gag codingsequence. Peak 3 overlaps the gag gene stop codon and the polgene start codon. Peaks 4–7 are within the pol coding sequence.Peak 8 is within the vif coding sequence and also overlaps with theexonic splicing enhancer for vpr (ESEVpr). Peak 9 overlaps with theexonic splicing silencer 2p (ESS2p), the exonic splicing silencer 2(ESS2) and vpr coding sequence. Peak 10 lies within the envcoding sequence. Peak 11 overlaps with the RRE region. Peak 12 iswithin the env and rev coding sequences and overlaps the tat genestop codon and the exonic splicing enhancer 3a (ESS3a). Peak 13lies upstream of the env gene stop codon and the nef genestart codon. Peak 14 is within the nef coding sequence. Thesefindings identify the topology of the m6A landscape in HIV-1RNA and indicate widespread methylation throughout the genome.

Next, we performed MeRIP-seq experiments to determine thedynamics of RNA methylation in the host cells after HIV-1 infec-tion and identified a set of 56 gene transcripts that were specificallymethylated upon viral infection (Supplementary Table 2 andSupplementary Fig. 3). We performed gene ontology (GO) analysisof the 56 genes and found that the most represented category was‘viral gene expression’, suggesting that methylation of many of thetranscripts is functionally important for viral infection (the topten most enriched categories are shown in SupplementaryFig. 3a). In fact, of the 56 genes, 19 have a known associationwith HIV replication and in some cases, the protein productsdirectly interact with viral components. Remarkably, the majorityof these genes encode proteins with proviral functions. Depletionor inhibition of MOGS, PSIP1 (LEDGF), HNRNPK, TRAF2,TUBA1B, CCT2, PABPC3, CBFB and ETS2 have been reported tonegatively affect different viral replication steps (SupplementaryTable 2 and references therein); expression of GTF2I, MYL12Aand UBA52 is induced by the infection process or by viral proteins;HLA-DPA2, EIF3M, DUT, MBD2 and RPS5 proteins have beenshown to interact with viral proteins; and HSPA1A and SRSF5overexpression is known to promote viral replication.

The exact mechanism by which m6A regulates the stability,splicing and translation of RNA transcripts remains unknown. Togain insight, we compared the distribution of m6A peaks in the56 HIV-1-specific methylated RNAs versus the total infected cellu-lar transcriptome and found some striking differences (Fig. 2b).Specifically, the percentage of total m6A peaks mapping to thehuman genome located in the 5′UTR, CDS, 3′UTR and intronicregions was 17.5%, 66.0%, 14.4% and 2.1% for the 56 HIV-specificm6A peaks, respectively, compared with 13.4%, 41.0%, 31.6% and14.0% for the same regions in the total cellular transcriptome ofinfected cells (Fig. 2b). Thus, for the HIV-1-specific methylatedgenes, m6A is more abundant in the 5′UTR and CDS and less abun-dant in the 3′UTR and intronic regions of HIV-1 RNA comparedwith total cellular mRNA. These findings suggest that m6A modifi-cation may have more prominent functions in RNA splicing and

translation than in RNA stabilization. Additionally, m6A appearsto be more prevalent on mature than immature mRNAs, as indi-cated by the large reduction in m6A peaks in the intronic regionof HIV-1 RNA versus total cellular mRNA.

RNA methylation is a dynamic process controlled by the catalyticactivities of the m6A writer (MTases) and eraser (demethylases)enzymes. Because the enzymes have preferred target sequences8,9,13,18,19,we performed consensus sequence motif analysis within m6A peaksto determine the preferred motifs in HIV and cellular RNA, anddetermined whether the preferential motif usage in the host cellswas altered by HIV-1 infection. In uninfected T cells, the frequencyof MGACK (A/C-GAC-G/U), RRACH (G/A-G/A-AC-A/C/U) andUGAC motifs within m6A peaks was ∼37%, 33%, and 30%, respect-ively (Fig. 2c). After HIV-1 infection, enrichment of m6A in motifMGACK was increased (from 37% to 42%), indicating a largechange in motif preference during viral infection. In HIV-1 RNA,UGAC and MGACK were equally preferred (∼37%), but UGACusage was ∼7% higher than in the cellular transcripts. Althoughthe mechanisms by which viral infection drives a change in the pre-ferred host m6A motif are not clear, several plausible possibilitiesexist. For example, the assembly and catalytic rate of the methylationcomplex may be changed by viral infection such that MGACKbecomes the preferred substrate. Conversely, infection-drivenchanges in demethylase activities may decrease recognition ofmethylated MGACK but not of the other sequence motifs. Or, theaccessibility of these motifs is altered in this context, either bysequestering or secondary structure. Future characterization ofmethylation complexes in HIV-1-infected cells will undoubtedlyenhance our understanding of the dynamics of mammalian andviral RNA methylation during host–virus interactions.

m6A methylation modulates the interaction between Rev proteinand RRE RNA. In considering the mechanism by which m6Amight alter RNA function to promote HIV-1 replication, wehypothesized that m6A might alter the secondary or tertiarystructures of RNA and subsequently its ability to interact withproteins. To test this, we examined binding of viral Rev proteinto RRE RNA. RRE is a structurally and functionally well-characterized RNA element within the HIV-1 env gene. Aftertranslation, Rev proteins are imported back into the nucleuswhere they assemble at the RRE to form active nuclear exportcomplexes that facilitate the transit of viral transcripts into thecytoplasm32–34. This is an essential step in viral replication.Intriguingly, we found that peak 11 of the HIV-1 methylationsites (Fig. 2a) spanned the stem IIB region of RRE RNA, whichcontains the high-affinity binding site for Rev (Fig. 3a). Thestem loop secondary structure of RRE is critical for Rev proteinbinding and its function in nuclear export of HIV-1 RNA32–34.The solution structure of a Rev peptide bound to stem loop IIBshowed that Rev binds in the major groove of the RRE in adistinctive binding pocket formed by two purine–purine basepairs35. Therefore, we reasoned that methylation of A7877 andA7883 in stem loop IIB (Fig. 3a) might affect the RRE affinityfor Rev and thus alter the function of the RNA–protein complex.

To test this possibility, we first determined whether METTL3/METTL14 or AlkBH5 knockdown in MT4 cells modulated themethylation status of RRE RNA and its interactions with Rev.MeRIP experiments coupled with RT-qPCR specific for the RREsequence first confirmed that the two MTases and the demethylaseare indeed able to methylate and demethylate RRE, respectively(Supplementary Fig. 4a). Silencing of METTL3/14 decreased theamount of RRE RNA by Me-RIP, while knockdown of AlkBH5enhanced RRE RNA isolated by Me-RIP (SupplementaryFig. 4a). To determine whether changes in RRE methylationaffect its affinity for Rev, we performed experiments in 293Tcells transiently expressing the HIV-1 construct pLAI2. Rev–RRE

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interactions were detected by immunoprecipitation of Rev followedby qPCR to quantify bound RRE RNA. We found that Rev–RREbinding was significantly reduced by silencing of METTL3/METTL14 and, conversely, significantly increased by AlkBH5knockdown (Fig. 3b). These results demonstrate that methylation

of RRE RNA enhanced the formation of Rev–RRE complexesin vivo. Analysis of the REV immunoprecipitates indicated thatthese effects were not due to differences in the efficiency of Revimmunoprecipitation between cells expressing the variousshRNAs (Supplementary Fig. 4c).

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Figure 2 | RNA methylation profiles of HIV-1 and HIV-1-infected T cells. a, Methylation peaks in the full-length HIV-1 RNA genome. Viral gene annotationis shown below. Poly(A) RNA from uninfected and HIV-1-infected MT4 cells was fragmented, subjected to RNA-seq and MeRIP-seq, and analysed asdescribed previously8. Grey shading corresponds to the total RNA-seq mapping distribution (MeRIP input) and the red signal corresponds to the MeRIP-seqmapping distribution. On the x axis, the dark blue boxes and numbers indicate the m6A peaks called by MeRIPPeR analysis and referred to in the text.b, Topology of m6A in the cellular and viral transcriptomes. Pie charts show the distribution of total RNA sequence (upper) and m6A peaks (bottom) in fourregions (5′UTR, CDS, 3′UTR and intronic) of transcripts from uninfected and HIV-1-infected MT4 cells. The m6A peak distribution in HIV-1-specific RNAs(56 genes) is also shown (bottom, right). c, Frequency of UGAC, RRACH and MGACK sequence motifs within m6A peaks in cellular RNAs from control andHIV-1-infected MT4 cells (Ctrl_cellular RNAs and HIV_cellular RNAs, respectively) and HIV-1 LAI RNA (HIV_HIV RNA).






Because these experiments were performed with 293T cellsexpressing full-length HIV-1, it is possible that other viral proteinsmay have influenced Rev–RRE binding. To address this concern, weperformed the same experiments with cells expressing FLAG-Revand the 66-nucleotide minimal RRE stem loop region (Fig. 3a).Consistent with the results in cells expressing the full-lengthHIV-1 RRE, we found that Rev binding to the minimal RREsequence was significantly decreased by METTL3/METTL14knockdown and increased by AlkBH5 knockdown(Supplementary Fig. 4b,c). These data strongly suggest that

modulation of A7877 and A7883 methylation in the RRE stemloop IIB region affects its binding to Rev protein.

Rev–RRE interactions and their function in nuclear export ofviral RNAs are required events in the HIV-1 replication cycle. Wequestioned whether the changes in the binding ability of Rev toRRE due to the alteration of RNA methylation could affect the func-tion of viral RNA export. We depleted METTL3/METTL14 orAlkBH5 in 293 cells by corresponding shRNAs followed by infec-tions with HIV-1 LAI virus. Next, we prepared cytoplasmic andnuclear fractions from these cells and quantified the RRE RNA dis-tribution in the cytoplasm and nucleus. Hypo-methylation of RREcaused by METTL3/METTL14 knockdown significantly decreasedviral RNA nuclear export, consistent with the observed reductionin replication rates. In contrast, hyper-methylation of RRE RNAsby AlkBH5 silencing was able to enhance nuclear export and viralreplication (Supplementary Fig. 4d and Fig. 1c).

To define which adenosine in the RRE hairpin structure ismethylated in vivo, we performed primer extension experimentsusing AMV RT and Tth DNA polymerase36. A7877 and A7883modifications were analysed by sequence-specific complementaryssDNA probes and enzymatic extensions. In contrast with AMVRT, Tth pol enzyme is sensitive to m6A RNA modification, resultingin markedly reduced ability to extend the DNA oligo. As shown inFig. 3c, Tth pol failed to extend the probes for both A7877 andA7883 sites, indicating that these A residues are methylatedin vivo. In control experiments using ribosomal RNA, Tth polextended oligos for A4189 (unmethylated site) and did not extendto A4190 (methylated site), consistent with previously reportedresults36. Together, these results show that both A in the RREhairpin are methylated in vivo. m6A methylation of RRE may

Table 1 | HIV m6A peaks.

Peak no. Position (nt) Features1 625–875 PBS, DIS, ψ2 1950–2075 gag CDS3 2150–2375 gag STOP, pol START4 3000–3150 pol CDS5 3175–3275 pol CDS6 3325–3475 pol CDS7 3845–4075 pol CDS8 5325–5450 Vif CDS, ESEVpr9 5850–5950 ESS2p, ESS2, vpr CDS10 7550–7650 env CDS11 7875–8000 RRE, env CDS12 8450–8700 env CDS, rev CDS, tat STOP, ESS3a13 8725–8875 env CDS, nef START14 9000–9100 nef CDS

Abbreviations: PBS, primary binding site; DIS, dimerization sequence; ψ, encapsidation sequence;CDS, coding sequence; STOP, stop codon; START, start codon; ESE, exonic splicing enhancer; ESS,exonic splicing silencer; RRE, Rev-responsive element.

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Figure 3 | m6A methylation modulates the interaction between Rev protein and RRE RNA. a, Secondary structure of the hairpin loop IIB of HIV-1 RRE.The two highlighted adenosines represent the methylation sites identified in peak 11 on Fig. 2a. Nucleotide numbering is based on the pLAI2 proviral vectorsequence, and the mutation frequency of the adenosines in 2,501 HIV-1 sequences is shown in parentheses. b, RRE methylation enhances Rev interactions.293T cells were depleted of METTL3, METTL14 or AlkBH5 and transfected with pLAI2 vector. RIP-qPCR experiments were performed by IP with control IgGor anti-Rev antibody followed by qPCR to analyse RRE RNA. Results are the mean ± s.e.m. of three independent experiments and are expressed as the foldenrichment of RRE RNA in anti-Rev versus control IgG IPs. P values were calculated by unpaired two-tailed Student’s t-test. c, RRE A7877 and A7883 aremethylated in vivo. Primers were designed to probe for m6A modification at these two sites in RRE. AMV and Tth primer extensions for A7877 and A7883were performed on 1 µg poly(A)-enriched RNA from HIV-1 infected MT4 cells. Extensions for 28S A4189 and 28S A4190 were performed on 10 µg totalRNA and served as negative and positive methylation sites, respectively.






influence Rev binding by at least two mechanisms: direct interactionof the methyl group with specific Rev residues and/or by inducingchanges in RRE folding and thus the secondary structure requiredfor Rev recognition. Further experiments will be necessary toclarify the mechanisms involved.

HIV-1 replication and RNA nuclear export are perturbed bymethylation of A7883 in the RRE bulge region. To determinewhether m6A methylation in RRE RNA might confer anevolutionary advantage for HIV-1, we analysed the mutationfrequency of A7877 and A7883 in 2,501 HIV-1 sequences isolatedfrom infected humans. For this, we downloaded aligned FASTAsequences from the HIV database and processed the alignmentsusing a custom Python program. At each position in the referenceHIV-1 HXB2 genome, we calculated the frequency of mutations(single nucleotide substitutions and insertions/deletions) using thealignment matrix for the 2,501 sequences (Supplementary Fig. 5).We found that the mutation frequencies at positions A7877 andA7883 were 5.44% and 0.28%, respectively, which are much lowerthan the mutation frequencies of other adenosine residues in theHIV-1 sequence or of the adenosines in the stem loop IIB regionof RRE (11.16 ± 0.30% s.e.m. and 8.99 ± 6.80% s.e.m., respectively;Supplementary Fig. 5). This analysis suggests that A7877 andA7883 in the RRE are conserved and likely to confer survival

benefit. Interestingly, A7883 is located immediately above the high-affinity Rev-binding internal bubble structure of RRE stem loop IIB(Fig. 3a)37,38. Collectively, these results demonstrate a significantrole for A7877 and A7883 in Rev–RRE binding and function.

To further validate the role of specific methylation at positionA7877 and A7883 in HIV-1 replication, we generated LAI mutantconstructs in the RRE region (Fig. 3a): U7871C+A7877G (Mut1),A7883G (Mut2) and the combination of both (Mut3). Themutant constructs were assessed for viral replication by transfecting293T cells with wild type (WT) or mutant pLAI2 plasmids (Fig. 4a).Mut1, which is designed to affect methylation at position A7877, didnot show relevant differences in terms of viral expression in NTC,METTL3/METTL14 or AlkBH5 knockdown conditions comparedto WT virus. On the other hand, Mut2 and Mut3, which aremutated at the bulge region of RRE A7883, show a dramaticdecrease in viral replication (∼10% compared to WT) and thiseffect is not modulated by METTL3/METTL14 or AlkBH5 knock-down, suggesting that mutation at A7883 is not only responsible forseverely attenuating viral fitness but is also a functionally criticalnucleotide modified by the cellular RNA methylation machineries.Furthermore, we analysed the nuclear export efficiency of themutant constructs and compared them with the WT virus(Fig. 4b). Mutations at the A7877 region (Mut1) did not perturbthe nuclear export efficiencies of viral transcripts, but mutationsat A7883 in the bulge region (Mut2 and Mut3) drastically affectednuclear export of viral RNA (Fig. 4b). Taken together, theseresults demonstrate that methylation at A7883 is critical for Revbinding, enhanced nuclear export and viral replication.

DiscussionBased on these studies, we propose a model for m6A methylation ofRRE RNA and its effects on HIV-1 replication (Fig. 5). Silencing ofMETTL3/METTL14 or AlkBH5 induces m6A hypomethylation orhypermethylation of HIV-1 RRE sites at positions A7877 andA7883, respectively. Rev protein preferentially interacts withmethylated RRE. Therefore, AlkBH5 silencing increases RRE RNAmethylation and thus promotes Rev binding, increases nuclearexport of viral RNA and, subsequently, viral replication. In contrast,METTL3/14 silencing reduces methylation of RRE RNA and sub-sequent Rev recruitment onto the RRE, thereby reducing exportof viral RNA and suppressing replication. Our results, showingthe significance of A7883 in Rev–RRE interactions, are further sup-ported by in vitro Rev–RRE binding and NMR studies. In vitroexperiments determined that efficient Rev binding required A7883in the RRE structure39. NMR structural studies have shown thatA7883 is flipped out from the stem region of RRE and directly inter-acts with theW45 of Rev through van derWaals contacts35. Notably,mutation of W45 inactivates Rev40. Recent studies have furtherhighlighted the significance of m6A RNA modification in regulatingthe dynamics of RNA structure and RNA–protein interactionsinvolved in biological mechanisms22,41–43.

In summary, this work highlights the significance of RNAmethylation in the context of HIV-1 infection in human T cells.The demonstration that viral infection significantly increases m6Aabundance in both viral and host-cell RNA provides an impetus tofurther investigate the role played by RNA methylation in HIV-1biology and in the mammalian response to viral infection. The top-ology of m6A methylation in HIV-1 RNAs shows unique featurescompared to cellular mRNAs. m6A is abundant across the full-length viral genome and is present in regulatory regions, codingsequences and structural regions, suggesting that multiple aspectsof viral RNA biology may be regulated by m6A, possibly through dis-tinct mechanisms. We found that the RNA methylation status posi-tively correlates with viral replication, revealing a previouslyunknown layer of the regulation of HIV-1 replication. Precise lociof methylation can be further investigated by single-molecule

P = 0.0054

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Figure 4 | HIV-1 replication and RNA nuclear export are perturbed bymethylation of A7883 in the RRE bulge region. a, WT or LAI HIV-1 mutantconstructs, U7871C+A7877G (Mut1), A7883G (Mut2) and the combinationof both (Mut3), were assessed for replication in 293T cells under METTL3/METTL14 or AlkBH5 knockdown conditions. Results are the mean ± s.e.m. ofthree independent experiments and P values were calculated by unpairedtwo-tailed Student’s t-test. b, Nuclear export of viral RNA was analysed bycytoplasmic and nuclear fractionation in 293T cells transfected with WT orthe mutant LAI constructs described in a. Viral RNA distribution in the twocompartments is relative to the viral RNA content from total cellular RNA.Results are the mean ± s.e.m. of three independent experiments. P valueswere calculated by unpaired two-tailed Student’s t-test.






technologies, which are just emerging44 and also enable fully-phasedepitranscriptomic characterization of viruses, thus revealing the co-occurrence of these dynamic m6A sites. Finally, our finding thatRev–RRE binding is regulated by MTases and demethylases suggeststhat the pathways could be exploited pharmacologically to disruptRev function. Overall, these results identify a new mechanism forthe control of HIV-1 replication and its interaction with the hostimmune system. These findings also suggest that other RNAviruses may similarly use the m6A RNA modification as a mechan-ism to regulate viral and host RNA metabolism and gene regulation.

MethodsFor further methods see Supplementary Information.

HIV-1 virus production and MT4 infection. HIV-1 LAI virus was produced bytransfection of 293T cells with the proviral pLAI2 plasmid (NIH AIDS ReagentProgram) at 3 µg per 106 cells using Lipofectamine 2000 (Invitrogen), according tothe manufacturer’s instructions. After 4 h incubation, the medium was replaced withDMEM supplemented with 10% fetal bovine serum (FBS). Two days aftertransfection, the virus-containing supernatant was collected and filtered. The humanT cell line MT4 was then infected with HIV-1 LAI virus (106 c.p.m. of reversetranscriptase (RT) activity per 106 cells) and 12 h later, the cells were centrifuged toremove the virus, washed with PBS and resuspended in RPMI/10% FBS for furtheranalysis. MT4 and 293T cells were obtained from ATCC and were tested for theabsence of mycoplasma.

m6A immunoblot analysis. Total RNA was extracted from uninfected or HIV-1-infected MT4 cells (at 3 days post-infection) with TRIzol reagent (Invitrogen).Aliquots of 5 µg total RNAwere separated on 1% agarose/0.4% formaldehyde gels in20 mM HEPES and 1.5 mM EDTA, pH 7.8. RNA was stained with ethidiumbromide and transferred overnight to nitrocellulose membranes in 10× SSC buffer(1.5 M NaCl, 150 mM sodium citrate). RNA was UV-crosslinked to membrane andblocked in 5% milk PBS Tween 0.1%. Rabbit polyclonal anti m6A antibody was usedat 1:1,000 dilution in PBS Tween 0.1% with incubation at room temperature for 1 h.Subsequent steps for m6A immunoblotting were performed according to previouslyreported protocols8. For m6A dot blotting, poly(A) RNA was purified with anmRNA Direct Kit (Ambion) and the remaining steps were as described above.Densitometric quantification of the m6A signal intensity was performed usingImageJ software (NIH).

m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) andanalysis. The quality of the RNA samples was assessed using an Agilent Bioanalyzer

RNA 6000 Nano kit to ensure all RNA integrity numbers were >9.0. For poly(A)RNA purification, Dynabeads Oligo(dT)25 (61005, Life Technologies) wereincubated with an aliquot of 75 μg RNA, washed and subjected to a secondincubation with the same RNA sample, according to the manufacturer’sinstructions. RNA was obtained by ethanol precipitation overnight, fragmentedusing Ambion RNA Fragmentation Reagents (AM8740, Life Technologies) and thenrepurified by ethanol precipitation overnight. An aliquot of fragmented RNA wasretained as a control for RNA sequencing.

For MeRIP, fragmented RNAwas denatured for 5 min at 75 °C, cooled on ice for2–3 min and incubated with the anti-m6A-coupled beads in 300 µl of 140 mM IPBuffer for 2 h at 4 °C. Unbound RNAwas removed by centrifugation and beads werewashed three times with 140 mM IP buffer and then incubated in elution buffer(5 mM Tris HCl, pH 7.5, 1 mM EDTA, 0.05% SDS, and proteinase K) for 1.5 h at50 °C. The eluted RNAwas extracted with phenol/chloroform and precipitated withethanol. An aliquot of RNA was retained as a control for the first round of MeRIPRNA, and the remainder was subjected to a second round of MeRIP, as describedabove. Libraries for sequencing (input RNA-Seq and MeRIP-Seq) were preparedusing Illumina TruSeq Stranded mRNA kits, entering the protocol at the Elute-Prime-Fragment step, with the modification that samples were heated to 80 °C for2 min to only prime but not further fragment. Samples were sequenced on a HiSeq2000 across two lanes with single read 50. RNA-seq data were aligned to a combinedhg19 (chr 1–22, X, Y) and HIV-1 (https://aidsreagent.org/, pLAI2) genome FASTAusing the STAR aligner45, keeping only uniquely mapping reads. Peaks were calledwith MeRIPPeR8, using Fisher’s Exact Test on 25 bp genome-based windows to testfor statistically significant enrichment in the IP relative to the control input RNA,with Benjamini–Hochberg P-value adjustment and a P-value cutoff of 0.05.Windows were merged into peaks, and peaks smaller than 100 bp were filtered out.Differentially methylated peaks were called by splitting the union of all peaks into100 bp windows in 25 bp steps. MeRIP-seq and RNA-seq counts were tabulatedusing bedtools46 and input into edgeR47 to identify windows with significant changesin methylation. Read counts mapping to genes were obtained using HTSeq(https://pypi.python.org/pypi/HTSeq), gene features using r-make count(http://physiology.med.cornell.edu/faculty/mason/lab/r-make/usage.html) and allother features using BedTools. RNA-Seq data are publicly available at GEO GSE74016.

Gene ontology (GO) analysis of the uniquely methylated transcripts identifiedduring HIV-1 infection was performed using AmiGO software. The top tencategories are shown.

Rev–RRE interaction experiments. For the Rev–RRE interaction experiments, wetransfected 3 µg pLAI2 plasmid per 106 293T cells. After 4 h, the medium wasreplaced with fresh DMEM/10% FBS and incubated for a further 44 h. Cells werethen lysed in IP lysis buffer (Pierce) and Rev protein was immunoprecipitated byincubation overnight at 4 °C with anti-Rev antibody48 (NIH AIDS Reagent Program)(5 µg/10 × 106 cell equivalents) and 50 µl protein A/G magnetic beads (Pierce). Foroverexpression of Rev protein, the Rev coding sequence was amplified from cDNA

RRE

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Figure 5 | Proposed model for modulation of the m6A modification in RRE RNA and its effect on HIV-1 replication. See text for details. For simplicity, onlymonomeric Rev–RRE interactions are shown.




https://aidsreagent.org/

https://pypi.python.org/pypi/HTSeq

http://physiology.med.cornell.edu/faculty/mason/lab/r-make/usage.html

http://www.pdb.org/pdb/search/structidSearch.do?structureId=GSE74016



isolated from HIV-1-infected MT4 cells and cloned into pFLAG-CMV-2 (Sigma)using EcoRI and SalI restriction sites. A minimal RRE corresponding to a66 nucleotide region (GCA CTA TGG GCG CAC GGT CAA TGA CGC TGA CGGTACAGG CCAGACAAT TAT TGT CTGGTATAG TGC) was PCR amplified andcloned into pcDNA3 (pcDNA3_minRRE) using HindIII and EcoRI restriction sites.The FLAG-Rev construct (1.5 µg) was co-transfected with pLAI2 (1.5 µg) orpcDNA3_minRRE (1.5 µg) into 106 293 T cells with Lipofectamine 2000. After 4 h,the medium was replaced with fresh DMEM/10% FBS and incubated for a further44 h. Cells were then lysed in IP lysis buffer (Pierce) and Rev protein wasimmunoprecipitated by incubation for 2 h at room temperature with anti-FLAGantibody (2 µg/3 × 106 cell equivalents) and 10 µl protein A/G magnetic beads(Pierce). IPs with mouse IgG (Santa Cruz Biotechnology) were performed in parallelas negative controls. In all the experiments, the beads were washed three times withIP lysis buffer and divided into equal volumes. One aliquot was extracted withTRIzol for RRE RNA quantification by qPCR, with normalization to β-actinRNA. The second aliquot was subjected to western blotting to assessanti-FLAG IP efficiency.

MeRIP-qPCR analysis of RRE methylation. MT4 cells (5 × 106) were transducedwith lentiviruses encoding METTL3, METTL14 or AlkBH5 shRNA as describedabove. After 3 days, cells were infected with LAI virus and RNA was extracted 48 hlater. Aliquots of 40 µg total RNA were poly(A) enriched using mRNA directisolation kit (Ambion) and enriched fractions were fragmented into 60–200nucleotides with RNA fragmentation reagents (Life Technologies AM8740).Fragmented RNA was incubated with 3 µg anti-m6A and 15 µl protein A/Gmagnetic beads for 2 h at room temperature in MeRIP buffer (10 mM Tris HCl, 150mM NaCl, 0.1% NP-40, pH 7.4). IPs with rabbit normal IgG (10500C, Invitrogen)were performed in parallel and served as input controls. Beads were washed threetimes with MeRIP buffer and m6A-containing RNAs were eluted by incubation with20 mM m6A for 20 min at room temperature. Eluted RNA fractions wereprecipitated using the sodium acetate/ethanol precipitation method and cDNAsynthesis and qPCR analysis were performed as described above.

AMV reverse transcriptase and Tth DNA polymerase primer extension analysesfor probing site-specific m6A modification

These experiments were performed as previously described36 with the followingmodifications. ssDNA oligos were 5′ radio-labelled with 32P. For 28S 4189 and 28S4190 (negative and positive controls) probing, 10 µg total RNA from HIV-1 infectedMT4 cells was used. For A7877 and A7883, 1 µg poly(A) RNA from HIV-1 infectedMT4 cells was used. RNA and radio-labelled DNA oligos (1 µl) were annealed in 2×annealing solution in a total volume of 15 µl with 1× Tth pol buffer with 1 mMMnCl2 (Promega) or AMV buffer (Promega). The mixture was denatured at 95 °Cfor 10 min and slowly annealed, cooling to room temperature. Annealed solution(3 µl) was mixed with 2 µl enzyme and heated at 37 °C (AMV ReverseTranscriptase) or 55 °C (Tth DNA Polymerase) for 2 min. dTTP solution (finaldTTP concentration, 100 µM) was added and the reactions were incubated for30 min at 37 °C (AMV) or at 55 °C (Tth). Reactions were resolved on a 20%denaturing polyacrylamide gel and exposed overnight on phosphoimaging screen.ssDNA probe sequences are listed in Supplementary Table 3.

Accession numbers. RNA-Seq data are publicly available at GEO under accessionnumber GSE74016.

Received 13 August 2015; accepted 22 January 2016;published 22 February 2016

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http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE74016



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AcknowledgementsThe authors thank S. Head and the staff of the Next Generation Sequencing core facility atthe Scripps Research Institute for help with the early phase of the project and HT-seq. Theauthors thank J. Klabis for help with the preparation of figures, and members of the Ranalaboratory for discussions and advice, especially T.-C. Chao and K. Chang. The authors

thank the Sloan Foundation (2015-13964), the Bert L. and N. Kuggie Vallee Foundation,the Irma T. Hirschl and Monique Weill-Caulier Charitable Trusts, the WorldQuantFoundation and the STARR Consortium (I7-A765, I9-A9-071) and acknowledge supportfrom the National Institutes of Health (EB020393, NS076465, C.E.M.; NIAID and NIDAAI43198, DA039562 and DA030199, T.M.R.).

Author contributionsG.L. designed and performed experiments, analysed data and wrote the paper. S.G.performed experiments and analysed data. Y.S. performed experiments and analysed data.G.M.G. performed experiments and analysed data. V.B., Y.W. and C.M. analysed data.T.M.R. contributed to the concept and design, data analysis and manuscript writing.

Additional informationSupplementary information is available online. Reprints and permissions information isavailable online atwww.nature.com/reprints. Correspondence and requests formaterials shouldbe addressed to T.M.R.

Competing interestsThe authors declare no competing financial interests.





http://www.nature.com/reprints



Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells

Gianluigi Lichinchi, Shang Gao, Yogesh Saletore, Gwendolyn Michelle Gonzalez, Vikas Bansal, Yinsheng Wang, Christopher Mason & Tariq M. Rana

Supplementarymethods,figuresandtables:

Supplementary Methods Supplementary methods

Supplementary Figure 1 m6A content of poly(A)-enriched RNA

Supplementary Figure 2 METTL3,METTL14,andAlkBH5knockdownefficiency

Supplementary Figure 3 56 genes are uniquely methylated upon HIV-1 infection

Supplementary Figure 4RREmethylationismodulatedbyMETTL3,METTL14,andAlkBH5proteinlevelsandaffectsnuclearexportofviralRNAs

Supplementary Figure 5 Frequency of adenosine mutation in HIV sequences

Supplementary Figure 6 Rawdataforblotsandgels

Supplementary Table 1 HIV-1 and human RNA sequence summary

Supplementary Table 2 HIV-1-specificm6Atranscripts

Supplementary Table 3 shRNA sequences

Supplementary Table 4 Primer sequences











































SUPPLEMENTARY INFORMATIONARTICLE NUMBER: 16011 | DOI: 10.1038/NMICROBIOL.2016.11



shRNA plasmids and lentiviral transduction

LentiviralparticlesforshRNA-mediatedgenesilencingwereproducedbytransfectionof293TcellswithpLKO(shRNA),psPAX2(packaging),andpMD2.G(envelope)plasmids(1:0.75:0.25ratio)usingLipofectamine2000.After2days,thesupernatantwascollected,filtered,mixedwith5µg/mlPolybrene,andaddedtoMT4cellsovernight.Thesupernatantwasthenremoved,andthecellswerewashedinPBSandresuspendedinfreshRPMI/10%FBS.Threedaysafterlentiviraltransduction,cellswereinfectedwithHIV-1asdescribedabove.Cellswereusedforexperimentsat3dayspost-infection.pLKOshRNAplasmidswerepurchasedfromSigma(MissionshRNALibrary).ShRNAsequencesarelistedinSupplementaryInformationTable3.

Western blot analysis

MT4cells(106/sample)werelysedinM-PERbuffer(Pierce),andsamplesof20µgtotalproteinwereresolvedby12%SDS-PAGEandtransferredtoPVDFmembranes(Bio-Rad).Membraneswereblockedfor1hatroomtemperaturewith2%BSAinPBST(PBS/0.1%Tween20),incubatedfor1hwithprimaryantibodies(1:1,000dilutioninBSA/PBST),washedwithPBST,andthenincubatedfor1hwithsecondaryantibodies(1:10,000dilutioninBSA/PBST).Antibodiesusedwere:rabbitmonoclonalanti-GAPDH(D16H11,CellSignalingTechnology),rabbitpolyclonalanti-METTL3(15073-1-AP,ProteintechGroup),rabbitpolyclonalanti-METTL14(HPA038002,Sigma),mousepolyclonalAlkBH5(SAB1407587,Sigma),rabbitanti-gagp24antiserum(65-005,BioAcademia),mousemonoclonalanti-Revantibody(1G7,NIHAIDSReagentProgram),mousemonoclonalanti-FLAGM2(F3165,Sigma),IRDye680-conjugatedanti-mouseIgG(LiCor),andIRDye800-conjugatedanti-rabbitIgG(LiCor).

Northern blot analysis

5′-amino-modifiedDNAoligonucleotidesspecificforHIV-1RNAwerepurchasedfromIDT.Theanti-HIV-1probesequenceswere: anti-gag:CCCGCTTAATACTGACGCTCTCGCACCCA, anti-vif:ATCCCTAATGATCTTTGCTTTTCTTCTTGGCACTACT, anti-nef:GCTCAGCTCGTCTCATTCTTTCCCTTACAGTAG.

OligonucleotideconjugationwithAlexaFluor680NHSesterwasperformedin0.1Msodiumtetraborate,pH8.5,for3hatroomtemperature.0.2mgofNHSesterwasdissolvedin30uLofDMSOand200uLofwater,mixedwith20ugofoligo.Theconjugationreactionwascarriedoutatrtfor3hrandthenblockedwithTris-buffer,pH7.5,finalconcentrationof50mMfollowedbyprecipitationwithsodiumacetateandethanol.RNAwasextractedwithTrizolreagentaccordingthemanufacturer’sinstructions.5µgtotalRNAwereloadedforeachlaneona1%agarose/0.4%formaldehydegelsin20mMHEPESand1.5mMEDTA,pH7.8.NorthernblottingwasperformedusingaNorthernMaxkit(Ambion).

RT-qPCR analysis

cDNAwaspreparedfrom1µgtotalRNAusingiScriptSupermix(Bio-Rad).qPCRreactionswereperformedusingSSoAdvancedSYBRGreenMix(Bio-Rad)withaLightCycler480instrument(Roche).QuantificationoftherelativefoldchangeinmRNAwasperformedbytheΔΔCpmethod.PCRprimersarelistedinSupplementaryInformationTable4.

Supplementary Methods

2 NATURE MICROBIOLOGY | www.nature.com/naturemicrobiology

SUPPLEMENTARY INFORMATION DOI: 10.1038/NMICROBIOL.2016.11


3.21680400

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Supplementary Figures

Supplementary Figure 1. m6A content of poly(A)-enriched RNA. Poly(A) RNA was isolated from uninfected (Ctrl) and HIV-1-infectedMT4cellswithanoligo(dT)-mRNAisolationkit.Enrichedfractionswerequantified,seriallydiluted,andimmu-noblottedwithanantibodyagainstm6A.Thedataisarepresentativeoffiveindependentexperiments.


SUPPLEMENTARY INFORMATIONDOI: 10.1038/NMICROBIOL.2016.11


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TL14mRNAlevel

M3/M14

shRNAs

M3 shR

NA

NTC shRNA

0.0

0.5

1.0

1.5

RelativeMET

TL3mRNAlevel

M3/M14

shRNAs

M3 shR

NA

NTC shRNA

Supplementary Figure 2.METTL3,METTL14,andAlkBH5knockdownefficiency.MT4cellswereinfectedwithlentivirusesencodingtheindicatedshRNAs,and3dayslater,knockdownefficiencywasassessedbywesternblot(left)andRT-qPCR(right)analyses.GAPDHservedasaloadingcontrolforwesternblotting.Thedataisarepresentativeoffiveindependentex-periments(n=3).RT-qPCRresultsarethemean±s.e.m.of5independentexperimentsandareexpressedasthemRNAlevelrelativetothatincellsexpressingnontargetingcontrol(NTC)shRNA.nindicatesthenumberoftechnicalreplicates.




b

a

Supplementary Figure 3. 56 genes are uniquely methylated upon HIV-1 infection. a, gene ontology analysis of the uniquely methylatedgenesduringHIV-1infection.The10mostenrichedcategoriesareshown.Fifty-sixgenesidentifiedtobeuniquelymethylatedduringHIV-1infectionareenrichedinvirus-relatedcategories.b,NormalizedreaddensitiesforGTF2I,PSIP1,SIAH2,STARD4,ETS2,DUTandABHD16AareshownforinputandMeRIPinuninfectedandHIV-infectedsamples.Thereaddensitiesareindicativeof2technicalreplicates.

15.0

12.5

10.07.55.02.50.0

RNA catabolicprocessTranslational termination

CellularproteincomplexdisassemblyViral transcription

mRNA metabolicprocessEstablishmentofproteinlocalizationtomembrane

mRNA catabolicprocessProteintargetingtomembrane

Multi-organismmetabolicprocessViral gene expression

-log(p-value)

Ctrl INPUT

Ctrl MeRIP

HIV MeRIP

HIV INPUT

m6A peak

Ctrl INPUT

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HIV MeRIP

HIV INPUT

m6A peak

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HIV MeRIP

HIV INPUT

m6A peak

Ctrl INPUT

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HIV MeRIP

HIV INPUT

m6A peak

STARD4SIAH2PSlP1GFT2I

Ctrl INPUT

Ctrl MeRIP

HIV MeRIP

HIV INPUT

m6A peak

Ctrl INPUT

Ctrl MeRIP

HIV MeRIP

HIV INPUT

m6A peak

Ctrl INPUT

Ctrl MeRIP

HIV MeRIP

HIV INPUT

m6A peaks

ABHD16ADUTETS2




dc

ba

Supplementary Figure 4.RREmethylationismodulatedbyMETTL3,METTL14,andAlkBH5proteinlevelsandaffectsnucle-arexportofviralRNAs.a,RRERNAmethylationlevelisdirectlymodulatedbymethyltransferaseanddemethylaseexpres-sion.MeRIP/RT-qPCRforRRERNAwasperformedinMT4cellsdepletedofMETTL3/METTL14orAlkBH5andsubsequentlyinfectedwithHIV-1LAIvirus.RNAwasextracted,poly(A)enriched,fragmented,immunoprecipitatedwithanti-m6Aantibody,andanalysedbyRT-qPCR.Resultsarethemean±s.e.m.ofthreeindependentexperiments(n=3)andareexpressedasthefoldenrichmentofminimalRRERNAinanti-FLAGversuscontrolIgGIPs.b,RIPexperimentswereperformedasdescribedinFig3b,exceptthat293Tcellswereco-transfectedwithFLAG-RevandpcDNA3_minRRE,whichencodestheminimalRRE.Results are the mean ± s.e.m. of of three independent experiments (n=3) and are expressed as the fold enrichment of minimal RRERNAinanti-FLAGversuscontrolIgGIPs.c,RIPefficiencywasassessedbywesternblotanalysisofinputmaterialandimmunoprecipitatedfractionsofpoly(A)-enrichedRNAfrom293Tcells.Top:RIPefficiencyanalysisfortheRevinpLAI2ex-pressionsystem.Bottom:RIPefficiencyanalysisfortheFLAG-RevandpcDNA3_minRREco-expressionsystem.Thedataisarepresentativeofthreeindependentexperiments.d,CytoplasmicandnuclearfractionationwasperformedonNTC,M3/M14andAlkBH5knock-down293Tcells.ViralRNAdistributioninthetwocompartmentsisrelativetotheviralRNAcontentfromtotalcellularRNA.Resultsarethemean±s.e.m.ofthreeindependentexperiments(n=4).P-valuesarecalculatedbyunpairedtwo-tailedStudent’st-test.nindicatesthenumberoftechnicalreplicates.

Anti-FLAGAlkB

H5 shR

NA

M3/M14

shRNAs

NTC shRNAs

Ctrl IgG

AlkBH5 s

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M3/M14

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HIV-1RNArelativenuclearexport

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hRNA

M3/M14

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p=0.0006

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AlkBH5 s

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IP

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eRIP(x10

6 )




A788

3A7787

stem-lo

op IIB

(RRE) A

sHIV As

RRE

0

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35

Mutationfrequency(%

)

Supplementary Figure 5.FrequencyofadenosinemutationinHIVsequences.Atotalof2,501sequencesofHIV-1isolatedfrominfectedpatientswerealignedtothereferenceHXB2HIVgenome,andthemutationfrequencywascalculatedforalladenosines(As).WedownloadedalignedFASTAsequencesfor2501HIVgenomesfromtheHIVdatabaseandprocessedthemusingacustomPythonprogram.ForeachpositioninthereferenceHXB2genome,thedifferencesinnucleotides(A,C,T,Gand–[gap])werecalculatedusingthealignmentmatrix.Theposition,length,andfrequencyofinsertion/deletioneventswere also determined using the gaps in the aligned FASTA sequences. Mutation frequencies for total HIV-1, RRE, stem loop IIBintheRRE,andtwospecificadenosinesatpositions7787and7883areshown.Resultsarethemean+s.d.of2,501sequences.




Supplementary Figure 6.Rawdataforblotsandgels.Rawdataforblotsandgelsincludedinthemanuscriptareshownandlabeledwithmolecularweightmarkersandcorrespondingfigurenumbersinthemanuscriptandsupplementarymaterial.

CorrespondingtoSupplementaryFigure2

957255

43342617

95725543

342617

95725543

342617

957255

43

34

957255

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9572

55

43

GAPDH

AlkBH5

METTL14

GAPDHGAPDH

METTL3

Corresponding to Supplementary Figure 4c

95725543342617

95725543342617

FLAGIgG LC

IgG HC

RevIgG LC

IgG HC

Corresponding to Figure 1c

95725543342617

p55p41

p24

95725543

342617

GAPDH

Corresponding to Figure 3c

probeprobe+1

28SA4189

- AMV Tth

probe

probe+1

28SA4190

- AMV Tth

probeprobe+1

RREA7877

- AMV Tth

probeprobe+1

RREA7883

- AMV Tth




Size Control_1_Control

Control_1_MeRIP_2_Rounds

Control_2_Control

Control_2_MeRIP_1_Round

HIV_1_Control

HIV_1_MeRIP_1_Round

HIV_2_Control

HIV_2_MeRIP_1_Round

chr1 249250621 6832868 3933593 3784733 2680944 2123715 1892236 2179434 2262238

chr2 243199373 6469023 3379318 3243756 2181020 1845529 1530029 1883793 1833678

chr3 198022430 4288317 2504991 2806320 1959027 1722190 1442884 1768307 1733797

chr4 191154276 1924022 1088681 1075035 695596 670717 516272 685510 616162

chr5 180915260 3304806 2053934 2030476 1467805 1294757 1107218 1326916 1327188

chr6 171115067 4426746 2962792 2867309 2089975 1756755 1589860 1803350 1909249

chr7 159138663 3614281 2393964 2404143 1766587 1279686 1136342 1337267 1380211

chr8 146364022 3289331 1779649 1920905 1345071 1112776 958414 1142973 1155069

chr9 141213431 3352335 2230637 2208497 1597142 1273979 1173074 1320443 1410187

chr10 135534747 2203088 1338180 1451100 1029882 849405 722414 878972 872825

chr11 135006516 5493788 4874352 3025129 2599619 1834762 1834245 1866841 2186417

chr12 133851895 4961434 2774654 2894508 1973728 1616271 1399844 1666060 1677010

chr13 115169878 1339693 692360 860444 596699 534065 404220 543055 488834

chr14 107349540 2390202 1504134 1584200 1157848 901800 796801 931435 960097

chr15 102531392 2808404 1809680 1865360 1381717 1032047 915998 1059474 1092762

chr16 90354753 4233523 2663532 2505213 1809370 1523854 1413499 1588488 1700736

chr17 81195210 4993594 3507752 3101901 2387111 1672225 1626389 1728790 1946162

chr18 78077248 666486 350022 429600 285581 270072 212524 276980 258260

chr19 59128983 6015744 4421623 3564544 2716342 1892420 1925482 1967896 2315191

chr20 63025520 1685301 1126442 1124684 829575 632996 586752 655208 706012

chr21 48129895 2495103 872403 804789 508790 454161 377432 458939 449236

chr22 51304566 2289959 1670515 1563640 1147658 832175 809748 870700 965505

chrX 155270560 2457491 1541016 1581668 1163641 869894 780667 896888 941493

chrY 59373566 303054 117952 95046 62774 55584 48807 56244 57087

chrM 16571 3131999 2128883 1730509 1549403 1867543 1155382 1658133 1366022

HIV 11738 40695 28682 12988 15171 29292810 18503288 28420526 23683111

Human 3095693983 84970592 53721059 50523509 36982905 29919378 26356533 30552096 31611428

HIV 11738 40695 28682 12988 15171 29292810 18503288 28420526 23683111

Total 85011287 53749741 50536497 36998076 59212188 44859821 58972622 55294539

Percentage

Human 99.95% 99.95% 99.97% 99.96% 50.53% 58.75% 51.81% 57.17%

HIV 0.05% 0.05% 0.03% 0.04% 49.47% 41.25% 48.19% 42.83%

Supplementary Table 1. HIV-1 and human RNA sequence summary

Supplementary Tables




Supplementary Table 2.HIV-1-specificm6Atranscripts

Yes:HIV-relatedgene,nofunctionaldataavailable.Yes(+):HIV-relatedgene,proviralfunction.Yes(+/-):HIV-relatedgene,uncertainfunction.

GeneSymbol Molecular Function PreviouslylinkedtoHIV-1 Reference(PMID)AKIRIN1 Cytoskeleton organizationPLK3 KinasePTRHD1 Peptydil-tRNA hydrolaseB3GNT2 GlucosaminyltransferaseMOGS Glucosidase Yes(+) 25318123DUSP2 PhosphataseMZT2B Cytoskeleton organizationTTN-AS1 Not knownRPL32 RibosomalproteinZBED2 DNAbindingFAM162A ApoptosisSIAH2 UbiquitinproteinligaseMFSD10 MembranetransporterGPR125 G-protein coupled receptorRPS23 RibosomalproteinCOX7C CytochromeCsubunitSTARD4 Sterol transportRPS14 RibosomalproteinABHD16A abhydrolaseHSPA1A Chaperone Yes(+/-) 21970979HLA-DPA1 MHC class II protein Yes 22860026GTF2I Transcription factor Yes(+) 21613400ZNHIT1 TranscriptionATP6V1F ATPaseRPL30 RibosomalproteinPSIP1 DNAbinding Yes(+) 24261564OSTF1 Stimulating factorHNRNPK Ribonucleoprotein Yes(+) 18854243TRAF2 TNF receptor-associated factor Yes(+) 23774506RPLP2 RibosomalproteinPRKCDBP PKCbindingproteinILK KinaseDENND5A GolgitraffickingEIF3M Translation Yes 22174317TUBA1B Cytoskeleton organization Yes(+) 19460752CCT2 Chaperone Yes(+) 18976975PABPC3 poly(A)-bindingprotein Yes(+) 19460752HAUS4 Cytoskeleton organizationSRSF5 Splicing factor Yes(+) 22933280C14orf1 Not knownDUT deoxyuridine triphosphatase Yes 22944692UBL7 Ubiquitin-likeproteinCOX5A CytochromeCsubunitNDE1 Not knownCBFB HIV Vif function Yes(+) 22190037APRT adeninephosphorybosiltransferaseRNASEK RNAseRNASEK-C17orf49 Not knownMYL12A myosin, regulatory Yes 19794400MBD2 methyl-CpGbindingprotein Yes 19454010UBA52 Ubiquitin-ribosomalprotein Yes 23874603RPS16 RibosomalproteinRPS5 Ribosomalprotein Yes 22174317MIR3648-1 Not knownETS2 Transcription factor Yes(+) 18854154MAGED2 p53 function




shRNA Sequence

Non targeting control (NTC) CCGCAGGTATGCACGCGT

METTL3-1 CCGGGCAAGTATGTTCACTATGAAACTCGAGTTTCATAGTGAACATACTTGCTTTTTG

METTL3-2 CCGGGCCAAGGAACAATCCATTGTTCTCGAGAACAATGGATTGTTCCTTGGCTTTTTG

METTL14-1 CCGGCCATGTACTTACAAGCCGATACTCGAGTATCGGCTTGTAAGTACATGGTTTTT

METTL14-2 CCGGGCCGTGGACGAGAAAGAAATACTCGAGTATTTCTTTCTCGTCCACGGCTTTTT

AlkBH5-1 CCGGGAAAGGCTGTTGGCATCAATACTCGAGTATTGATGCCAACAGCCTTTCTTTTTG

AlkBH5-2 CCGGCCACCCAGCTATGCTTCAGATCTCGAGATCTGAAGCATAGCTGGGTGGTTTTTG

Supplementary Table 3. shRNA sequences




Primer Sequence Use

GAPDHFwd TGGCGGGGAAGTCAG qPCR

GAPDHRev CGGAGGAGAAATCGGGC qPCR

gp120Fwd TGAGCCAATTCCCATACATTAT qPCR

gp120Rev CCTGTTCCATTGAACGTCTTAT qPCR

METTL3 Fwd GACACGTGGAGCTCTATCCA qPCR

METTL3Rev GGAAGGTTGGAGACAATGCT qPCR

METTL14 Fwd TCCCAAATCTAAATCTGACCG qPCR

METTL14Rev CTCTAAAGCCACCTCTTTCTC qPCR

AlkBH5 Fwd AGGGACCCTGCTCTGAAAC qPCR

AlkBH5Rev TCCTTGTCCATCTCCAGGAT qPCR

RRE Fwd GAGCAGCAGGAAGCACTATG qPCR

RRERev CCTCAATAGCCCTCAGCAA qPCR

β-actinFwd CACTCTTCCAGCCTTCCTTC qPCR

β-actinRev GGATGTCCACGTCACACTTC qPCR

28S4189 AGCTCGCCTTAGGACACCTGCGT AMV/Tthextension

28S4190 GAGCTCGCCTTAGGACACCTGCG AMV/Tthextension

A7877 AGACAATAATTGTCTGGCCTGTACCGTCAGCG AMV/Tthextension

A7883 TGCTGCACTATATCAGACAATAATTGTCTGGCCTGTACCG AMV/Tthextension

Mut1 Fwd ACTATGGGCGCACGGCCAATGGCGCTGACGGTACAGGCCAGA mutagenesis

Mut1Rev TCTGGCCTGTACCGTCAGCGCCATTGGCCGTGCGCCCATAGT mutagenesis

Mut2Fwd GGCGCACGGTCAATGACGCTGGCGGTACAGGCCAGACAATTAT mutagenesis

Mut2Rev ATAATTGTCTGGCCTGTACCGCCAGCGTCATTGACCGTGCGCC mutagenesis

Mut3 Fwd CACTATGGGCGCACGGCCAATGGCGCTGGCGGTACAGGCCAGACAATTA mutagenesis

Mut3Rev TAATTGTCTGGCCTGTACCGCCAGCGCCATTGGCCGTGCGCCCATAGTG mutagenesis

Supplementary Table 4. Primer sequences




dynamics of the human and viral m 6 a rna methylomes during … · 2017-12-23 · dynamics of the...

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