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N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

Summary The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification, as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A methyltransferases or an m6A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determined that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Altogether, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

Values are presented as mean ± SD (n=3), with ≥5000 cells counted per condition. *p≤0.05, ***p≤0.001 by two-way ANOVA with Bonferroni correction. (B) Viability of HCV-infected Huh7 cells treated with indicated siRNAs as measured at 72 hpi by Cell-Titer GLO assay. Data are presented as mean ± SD (n=3). (C) Immunoblot analysis of isolated nuclear (Nuc) and cytoplasmic (Cyto) biochemical fractions, as well as whole cell lysate (WCL), from extracts of mock-(-) or HCV-infected (+) Huh7 cells. (Lamin B -nuclear marker; Tubulin -cytoplasmic marker). (D) Quantification of protein levels in WCL (relative to Tubulin), or isolated nuclear (relative to Lamin B) and cytoplasmic (relative to Tubulin) fractions from extracts of mock-(-) or HCV-infected (+) Huh7 cells. Data are presented as the mean ± SEM of three independent experiments. (E) Specific infectivity of HCV particles produced by Huh7 cells treated with the indicated siRNAs. Specific infectivity was calculated as a ratio of HCV titer over HCV RNA isolated from the supernatant, measured at 72 hpi. Data are presented as the mean ± SEM of three independent experiments. (F) Focus forming assay of supernatants harvested from Huh7 cells at 72 hpi after treatment with the indicated siRNAs. Data are presented as the mean ± SEM of three independent experiments. (Right panel) Representative immunoblot analysis of extracts of HCV-infected Huh7 cells treated with the indicated siRNAs. (G) Parental Huh7.5 cells or Huh7.5 CD81 KO cells were stained with negative isotype control or CD81-specific antibodies and analyzed by fluorescence associated cell sorting. (H) Cells in (G) were infected with Gaussia luciferase expressing HCV Jc1 virus (orange bars) or vesicular stomatitis virus glycoprotein bearing lentiviral particles (VSVGpp) (purple bars) at an MOI of 0.05. Luciferase values are expressed as relative luciferase units (RLU). Data are presented as mean ± SEM of two independent experiments, each performed in triplicate (I) Naïve Huh7.5 cells were infected with supernatants from the HCV infections of the infections shown in panel (H). (J) RT-qPCR analysis of the indicated interferon stimulated genes relative to the housekeeping gene RPL30 from RNA extracted from Huh7 cells treated with the indicated siRNAs at 48 hours post HCV-infection. (K) RT-qPCR analysis of IFITM1 expression relative to RPL30 from RNA extracted from Huh7 cells treated with the indicated siRNAs at 24 hpi. Data in (J) and (K) are presented as mean ± SEM of two independent experiments performed in triplicate. *p≤0.05, ***p≤0.001 by unpaired Student's t-test.  Confocal micrographs of mock-or HCV-infected Huh7 cells expressing the indicated FLAG-tagged YTHDF proteins, immunostained with anti-FLAG (green) and anti-HCV core (red). Lipid droplets (grey) and nuclei (blue) were labeled with BODIPY and DAPI, respectively. (B) Huh7 cells stably replicating an HCV subgenomic replicon (Genotype 1B, K2040), and expressing the indicated FLAG-tagged YTHDF proteins and stained as in (A). Single cell images are representative of >30 cells analyzed. Scale bar, 10 µm.  https://euhcvdb.ibcp.fr/euHCVdb/jsp/nomen_tab1.jsp) representing all HCV genotypes ("Consensus DRACH") had lower polymorphism scores than DRACH sites found in the JFH1 reference strain but not the consensus ("JHF1 DRACH"). The A residue was particularly low scoring (p=3.00x10 -11 ) in a t-test between Consensus DRACH and JHF1 DRACH, suggestive of conserved methylation. Scores for the R, C, and H residues were also significantly lower (p=3.95x10 -5 , 2.84x10 -5 , and 0.02, respectively). The set of four DRACH motif sites proximal to the E1 m 6 A peak we identified from MeRIP-seq data ("E1 m 6 A peak sites") showed generally lower scores than the JFH1 DRACH, although only the R and H positions were statistically significant (p=0.02 and p=0.04). A control set of all other DRACH motifs found in the E1 region of the reference strain ("Other E1 DRACH") showed no significant differences in SNP rates with the JFH1 DRACH. We calculated polymorphism scores after a multiple sequence alignment using MUSCLE, where the score per position = -100*sum(p*log(p)), and p = frequency of each A, C, G, or T as described by ViPR (http://www.viprbrc.org). "Consensus" indicates that each position in the alignment contains a base consistent with the motif in > 50% of sequences. Statistical significance was determined by Student's t-test. (B) Focus-forming assay of extracellular and intracellular virus harvested from Huh7 cells after electroporation of WT or E1 mut HCV RNA (48h). Data are analyzed as the percent of viral titer relative to WT, and are presented as the mean ± SEM from at least four independent experiments. (C) Viral RNA in the supernatant harvested from Huh7 cells after treatment with the indicated siRNAs and infection with WT or E1 mut HCV, as measured by RT-qPCR. Data are presented as the mean ± SD, and are representative of two experiments done in triplicate. *p≤0.05, **≤0.01, p***p≤0.001 by unpaired Student's t-test relative to siCTRL-WT sample.
For CRISPR-mediated gene knockout, we generated two expression plasmids encoding U6 promoter driven human CD81-specific guide RNAs (gRNAs) (Mali et al., 2013). Two rounds of overlapping PCR were performed by amplifying a gRNA encoding plasmid (provided by George Church, Harvard University; Addgene plasmid # 41819): in the first round, PCR products were generated encompassing the U6 promoter through the 5′ end of the gRNA (consisting of the specific target sequence) with the ME-O-1122 oligonucleotide (5′-CGGGCCCCCCCTCGAGTGTACAAAAAAGCAGGCT) and a CD81 target sequence specific reverse oligonucleotide (see list below). A second PCR product was generated encompassing a region from the CD81 target sequence through the end of the gRNA coding sequence with a forward direction CLDN1 target sequence specific oligonucleotide (ME-O-1138; 5'-GCTTCATTCTCGCCTTCCGTTTTAGAGCTAGAAATA). These products were then reamplified with only the outer oligonucleotides, ME-O-1122 and -1123, to produce single PCR products flanked by XhoI and EcoRI sites at the 5′ and 3′ ends, respectively, and cloned into pBlueScript. Two separate CD81-specific gRNA plasmids were created with the following forward and reverse oligonucleotide combination: ME-O-1252/1251; 5'-GGGCTGCTACGGGGCCATC CGGTGTTTCGTCCTTTCC / 5'-GATGGCCCCGTAGCAGCCC GTTTTAGAGCTAGAAATA, and ME-O-1254/1253; 5'-TCTCGTGGAAGGTCTTCAC CGGTGTTTCGTCCTTTCC / 5'-CACCTGTACACGTAGGGCC GTGAAGACCTTCCACGAGA GTTTTAGAGCTAGAAATA, which target nucleotides 467-485 and 672-690 of the CD81 human cDNA (Genbank accession number: NM_004356), respectively. The sequences of oligonucleotide primers and plasmids used are available upon request.
In vitro transcription of HCV RNA and electroporation. Plasmid DNA encoding the described HCV constructs was linearized using XbaI. Linearized DNA treated with proteinase K (Thermo-Fisher) and Mung bean nuclease (NEB) was purified by phenol-chloroform extraction and was used as a template for in vitro transcription with a MEGAscript T7 Transcription Kit (Thermo-Fisher). Following DNase treatment, RNA was purified by phenolchloroform extraction and run on a denaturing gel to ensure integrity. For electroporation, 5µg of in vitro transcribed viral RNA was electroporated into 4x10 6 Huh7.5 cells at 250V and 950µF with a Gene Pulser Xcell system (Bio-Rad).
Luciferase assays for HCV Replication. Huh7.5 CD81 KO cells cultured in 12-well plates were transfected with specific siRNAs. At 24 hours after siRNA treatment, 1µg of in vitro transcribed JFH1-QL/GLuc2A reporter HCV RNA was transfected into cells using the TransIT-mRNA transfection kit (Mirus Bio), according to the manufacturer's instructions. Gaussia luciferase activity in supernatant harvested at 4, 24, 48 and 72 hours posttransfection was measured using the BioLux Gaussia Luciferase assay kit (NEB).
Quantification of RNA by RT-qPCR. RNA was reversed transcribed using the iScript cDNA synthesis kit (Bio-Rad) as per manufacturer's instructions, and synthesized cDNA was diluted 1:5 in water. RT-PCR was performed in triplicate with the Power SYBR Green PCR master mix (Thermo-Fisher) using the Applied Biosystems Step One Plus RT-PCR system. The sequences of primers used for RT-qPCR are:

Primer Name
Primer Sequence To measure extracellular HCV RNA, viral RNA from filtered cell supernatants was extracted using a QIAamp viral RNA kit (Qiagen), as recommended by the manufacturer. HCV RNA copy number was measured in triplicate by RT-qPCR using the TaqMan Fast Virus 1-Step Mix (Qiagen) with an HCV-specific probe targeting the 5' untranslated region of HCV (Assay ID: Pa03453408_s1). The copy number of HCV was calculated by comparison to a standard curve of a full-length in vitro transcribed HCV RNA, as described (Aligeti et al., 2015).
The enrichment index of YTHDF proteins measures the enrichment of YTHDF staining in the area proximal to lipid droplets, relative to that in the area distal to lipid droplets. This was calculated by using NIH Fiji/ImageJ to measure the mean brightness of YTHDF proteins in an area of 0.4µM around lipid droplets, as identified by BODIPY staining. Then, the YTHDF mean brightness in an area around the lipid droplets expanded by a further 0.4µM (between 0.4µM and 0.8µM) was subtracted from the first value.

RNA-immunoprecipitation.
Cell extracts were harvested in polysome lysis buffer (100mM KCl, 5mM MgCl 2 , 10mM HEPES (pH 7.0), 0.5% Nonidet P-40 (NP-40)) supplemented with protease inhibitor cocktail (Sigma) and RNaseIN ribonuclease inhibitor (Promega), and lysates were cleared by centrifugation. RNP complexes were immunoprecipitated with anti-FLAG antibody conjugated to magnetic beads (Sigma) overnight at 4°C with headover-tail rotation, and then washed five times in ice-cold NT2 buffer (50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM MgCl 2 , 0.05% NP-40). Protein for immunoblotting was eluted from ten percent of beads by boiling in 2X Laemmli sample buffer (Bio-Rad). RNA was extracted from ninety percent of beads using TRIzol reagent (Thermo-Fisher). Equal volumes of eluted RNA were used for cDNA synthesis, quantified by RT-qPCR and normalized to RNA levels in input samples. For HCV Core RIP, a similar procedure was employed except that RNP complexes were purified with Protein G Dynabeads conjugated to anti-Core.
For immunoprecipitation of reporter RNAs by FLAG-YTHDF2, Huh7 cells were co-transfected with WT or E1 mut psiCHECK2 plasmids along with plasmid expressing FLAG-YTHDF2 or vector control for 48 hours and RNA-immunoprecipitation was performed on extracts as described above.
Generation of Huh7.5 CD81 KO cells by CRISPR. Huh7.5 cells were transiently transfected with expression plasmids encoding a human codon optimized Cas9 protein from Streptococcus pyogenes (provided by G. Church, Harvard, Addgene plasmid # 41815) (Mali et al., 2013) and a pooled mixture of the CD81 gRNA expression plasmids (pSgCD81_1 and pSgCD81_2). Transfected cells were passaged for one to two weeks to allow the turnover of previously translated target protein. Cells were then FACS sorted for loss of CD81 following staining with the anti-CD81 antibody (mAb JS81, BD Pharmingen) and a goat anti-human Alexa-647 antibody (Thermo-Fisher). Knockout efficiency prior to sorting was between 7-8%. CD81 negative cells were then FACS sorted following staining with the same antibodies, and single cell clones were derived by dilution cloning in 96-well plates. Individual clones were expanded and assayed for CD81 expression by FACS staining, and their capacity to support HCV infection by limited dilution assay based on NS5A staining, as previously described (Meuleman et al., 2012). We chose Huh7.5 CD81 KO clone 5 as a representative single cell clone for further experiments. These cells, like all CD81 KO cells examined, lacked CD81 expression (Fig. S1G) and could neither be infected with (Fig. S1H) or subsequently produce infectious HCV (Fig. S1I).

PAR-CLIP.
FLAG-YTHDF or FLAG-GFP Huh7 cells lines were generated using pLEX-based lentiviral vectors as described (Kennedy et al., 2016), followed by clonal isolation. 6x10 7 cells from each cell line were seeded and the next day infected with HCV (MOI 0.2). 3 hpi, cells were pulsed with 100µM 4SU in fresh media. 48 hpi, cells were cross-linked (UV 365nm), harvested and PAR-CLIP was performed as described (Hafner et al., 2010;Kennedy et al., 2016). PAR-CLIP libraries were sequenced on a HiSeq 2000, base calling was performed with CASAVA, and data was then processed with the fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Reads with a length greater than 15 bp were used for downstream bioinformatic analysis. All alignments were performed with Bowtie (Langmead et al., 2009). Reads were initially aligned to the human genome build hg19 allowing up to 1 mismatch, and unaligned reads were then aligned to the HCV genotype 2A JFH1 cell culture-adapted viral genome (Aligeti et al., 2015) allowing 2 mismatches. These reads were then analyzed with PARalyzer (Corcoran et al., 2011) with a minimum cluster read depth of 3; the set of FLAG-GFP clusters (background) were then subtracted from the FLAG-YTHDF clusters with in house Perl scripts to yield the final dataset.
Viral RNA purification and LC-MS. 100µg total RNA from HCV-infected Huh7 cells was mixed with 200pmol each of five 5'-biotinylated oligonucleotides which were antisense to HCV RNA (Integrated DNA Technologies) in a total volume of 120µl, heated to 90°C for 5 minutes, and allowed to cool to room temperature. Oligonucleotide sequences are : 5' -GGTGCACGGTCTACGAGACCTCCC, 5' -CACGGACCTTTCACAGCTAGCCGT, 5' -GGGATCTCACCCTCCCGCCCGAGG, 5' -TAACGATGTCTATGATGACCTC, 5' -CAGAAGGATGACAATGACCTTC). Streptavidin MyOne C1 beads (100µl; Thermo-Fisher) were washed as recommended by the manufacturer, resuspended in 120µl 2X B&W buffer (10mM Tris-HCV, 1mM EDTA, 2M NaCl), and added to RNA/oligo annealing solution. After head-over-tail rotation at room temperature for 1 hr, beads were washed five times with 1X B&W buffer, bound viral RNA was eluted with 100mM DTT, and cleaned on Qiagen RNeasy mini columns using the manufacturer's protocol. Viral RNA enrichment was confirmed by RT-qPCR. Nucleosides were generated from 200ng purified RNA as previously described (Dominissini et al., 2016). Briefly, digestion was performed with nuclease P1 (Sigma, 2U) in buffer containing 25mM NaCl and 2.5mM ZnCl2 for 2h at 37⁰C, followed by incubation with Antarctic Phosphatase (NEB, 5U) for an additional 2h at 37⁰C. Nucleosides were separated and quantified using UPLC-MS/MS as previously described [Basanta-Sanchez et al.], except acetic acid was used in place of formic acid (Basanta-Sanchez et al., 2016).