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N6 -Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

N6 -Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection Article N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection Graphical Abstract Authors Nandan S. Gokhale, Alexa B.R. McIntyre, Michael J. McFadden, ..., Shelton S. Bradrick, Christopher E. Mason, Stacy M. Horner Correspondence [email protected] (C.E.M.), [email protected] (S.M.H.) In Brief N6-methyladenosine (m A) post- transcriptionally regulates RNA function. Gokhale et al. demonstrate that the RNA genomes of HCV, ZIKV, DENV, YFV, and WNV are modified by m A. Depletion of cellular machinery that regulates m Aor introduction of m A-abrogating mutations within HCV RNA increase viral particle production, suggesting that m A negatively regulates HCV. Highlights Accession Numbers d The RNA genomes of HCV, ZIKV, DENV, YFV, and WNV GSE83438 contain m A modification d The cellular m A machinery regulates HCV infectious particle production d YTHDF proteins reduce HCV particle production and localize at viral assembly sites d m A-abrogating mutations in HCV E1 increase infectious particle production Gokhale et al., 2016, Cell Host & Microbe 20, 654–665 November 9, 2016 ª 2016 The Authors. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.chom.2016.09.015 Cell Host & Microbe Article N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection 1 3,12 1 1 1 Nandan S. Gokhale, Alexa B.R. McIntyre, Michael J. McFadden, Allison E. Roder, Edward M. Kennedy, 3 4 5,6 1 1 7 Jorge A. Gandara, Sharon E. Hopcraft, Kendra M. Quicke, Christine Vazquez, Jason Willer, Olga R. Ilkayeva, 2 2 8,11 4 5,6 Brittany A. Law, Christopher L. Holley, Mariano A. Garcia-Blanco, Matthew J. Evans, Mehul S. Suthar, 8 3,9,10,14, 1,2,13,15, Shelton S. Bradrick, Christopher E. Mason, * and Stacy M. Horner * Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10021, USA Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA 30329, USA Duke Molecular Physiology Institute, Duke University, Durham NC 27701, USA Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY 10021, USA The Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY 10021, USA Programme in Emerging Infectious Disease, Duke-NUS Medical School, Singapore 169857, Singapore Tri-Institutional Program in Computational Biology and Medicine, New York City, NY 10065, USA Lead Contact Twitter: @mason_lab Twitter: @thehornerlab *Correspondence: [email protected] (C.E.M.), [email protected] (S.M.H.) http://dx.doi.org/10.1016/j.chom.2016.09.015 SUMMARY tions, N6-methyladenosine (m A) is the most abundant internal modification of eukaryotic mRNAs, contributing to RNA struc- The RNA modification N6-methyladenosine (m A) ture, localization, and function (Fu et al., 2014; Meyer and Jaf- frey, 2014). m A regulates many biological processes, including post-transcriptionally regulates RNA function. The stress responses, fertility, stem cell differentiation, circadian cellular machinery that controls m A includes methyl- rhythms, microRNA (miRNA) biogenesis, and cancer (Li and transferases and demethylases that add or remove Mason, 2014; Saletore et al., 2012; Yue et al., 2015; Zhou this modification, as well as m A-binding YTHDF pro- et al., 2015). However, little is known about its effects on micro- teins that promote the translation or degradation of bial infection. m A has long been known to be present in the 6 6 m A-modified mRNA.We demonstratethat m A mod- RNA transcripts of viruses with nuclear replication, such as influ- ulates infection by hepatitis C virus (HCV). Depletion enza A virus, simian virus 40, Rous sarcoma virus, avian sar- 6 6 of m A methyltransferases or an m A demethylase, coma virus, and adenovirus (Dimock and Stoltzfus, 1977; respectively, increases or decreases infectious HCV Kane and Beemon, 1985; Krug et al., 1976; Lavi and Shatkin, particle production. During HCV infection, YTHDF 1975; Sommer et al., 1976). More recently, we and others proteins relocalize to lipid droplets, sites of viral as- have shown that m A serves as a positive regulator of HIV-1, sembly, and their depletion increases infectious viral a retrovirus with a nuclear replication step (Kennedy et al., 2016; Lichinchi et al., 2016; Tirumuru et al., 2016). However, a particles. We further mapped m A sites across the role for m A in regulating the life cycle of viruses that replicate HCV genome and determined that inactivating m A exclusively in the cytoplasm, such as viruses within the Flavivir- in one viral genomic region increases viral titer without idae family, has been unexplored. Flaviviridae, including Zika affecting RNA replication. Additional mapping of m A virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), yellow on the RNA genomes of other Flaviviridae, including fever virus (YFV), and hepatitis C virus (HCV), represent both dengue, Zika, yellow fever, and West Nile virus, iden- established and emerging pathogens. They contain a positive- tifies conserved regions modified by m A. Altogether, sense, single-stranded RNA genome that encodes a viral this work identifies m A as a conserved regulatory polyprotein and use similar replication strategies. RNA-based mark across Flaviviridae genomes. regulation of these viral genomes plays a fundamental role in their infection, such as the liver-specific miRNA miR-122 for HCV replication, RNA structural elements for HCV and DENV 0 0 INTRODUCTION replication, and 2 -O methylation of the 5 cap of WNV RNA for immune evasion and WNV replication (Bidet and Garcia- The chemical modification of RNA is an important post-tran- Blanco, 2014; Hyde et al., 2014; Jopling et al., 2005; Mauger scriptional regulator of RNA. Of the many known RNA modifica- et al., 2015; Pirakitikulr et al., 2016). 654 Cell Host & Microbe 20, 654–665, November 9, 2016 ª 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 6 The cellular machinery that regulates m A includes proteins and FTO were unaffected (Figures S1C and S1D). Thus, the 6 6 that act as writers, erasers, and readers of m A. The addition m A methyltransferases negatively regulate HCV infection, while 6 m 6 of m A on mRNA, which occurs at the consensus motif DRA CH the m A demethylase positively regulates HCV infection. (where D = G/A/U, R = G > A, and H = U/C/A), is mediated by a We next defined the stage of the HCV life cycle regulated by methyltransferase complex containing the methyltransferase- the m A machinery. Depletion of METTL3+14 significantly like (METTL) enzymes METTL3 and METTL14 and the cofactors increased the production of infectious virus and viral RNA in Wilms tumor 1-associated protein (WTAP) and KIAA1429 (Fu the supernatant compared to control siRNA at 72 hpi (Figures et al., 2014; Liu et al., 2014; Meyer and Jaffrey, 2014; Schwartz 1D and 1E). Conversely, depletion of FTO decreased infectious et al., 2014; Yue et al., 2015). The removal of m A from mRNA virus and HCV RNA in the supernatant (Figures 1D and 1E) is catalyzed by the demethylases fat mass and obesity-associ- without altering the viral-specific infectivity (Figure S1E). Deple- ated protein (FTO) or a-ketoglutarate-dependent dioxygenase tion of ALKBH5 did not affect viral titer or protein levels, indi- AlkB homolog 5 (ALKBH5) (Jia et al., 2011; Zheng et al., 2013). cating that this demethylase does not influence the HCV life The cytoplasmic YTH-domain family 1 (YTHDF1), YTHDF2, and cycle (Figure S1F). We next tested whether the altered HCV titer 6 6 YTHDF3 proteins bind to m A through their C-terminal YTH after m A machinery depletion was due to altered viral RNA repli- domain. Functionally, YTHDF1 promotes the translation of cation. In these experiments, we used Huh7.5 CD81 knockout 6 6 m A-modified mRNA, while YTHDF2 targets m A-modified (KO) cells, in which essential HCV entry factor CD81 (Zhang mRNAs for degradation (Wang et al., 2014, 2015). The function et al., 2004) was deleted by clustered regularly interspaced short of YTHDF3 is still unknown. The discovery of these proteins palindromic repeats/Cas9 (CRISPR/Cas9), resulting in cells and the development of high-throughput m A-mapping tech- permissive for HCV RNA replication and viral particle production niques have led to many insights into the function of m A(Dom- following viral RNA transfection that are unable to support sub- inissini et al., 2012; Fu et al., 2014; Linder et al., 2015; Meyer sequent rounds of viral infection (Figures S1G–S1I). In these et al., 2012). Nonetheless, many aspects of the regulation of spe- cells, we depleted METTL3+14 or FTO by siRNA, transfected cific mRNAs by m A remain unexplored. the cells with in vitro transcribed RNA of the HCV reporter virus Here, we define a role for m A in regulating the life cycle JFH1-QL/GLuc2A, and measured HCV RNA replication by as- of HCV. We demonstrate that the m A methyltransferases saying for secreted Gaussia luciferase (Yamane et al., 2014). negatively regulate the production of infectious HCV particles Depletion of METTL3+14 or FTO had no effect on Gaussia lucif- and that the m A-binding YTHDF proteins all relocalize to sites erase levels compared to control over the time course, while our of HCV particle production and suppress this stage of viral negative control RNA containing a point mutation in the viral infection. We map m A across the HCV RNA genome and RNA-dependent RNA polymerase (Pol ) did not replicate (Fig- 6 6 show that preventing m A at one of these regions enhances ure 1F). These data indicate that m A dynamics do not regulate viral titer by increasing the interaction of the HCV RNA with HCV translation or RNA replication but do regulate the produc- the HCV Core protein. Finally, we describe viral RNA m A-epi- tion or release of infectious viral particles. transcriptomic maps for several other Flaviviridae, including Changes in expression of the m A machinery have been ZIKV, DENV, WNV, and YFV. Altogether, our data reveal that shown to affect cellular gene expression (Dominissini et al., m A regulates HCV infection and set the stage for the explora- 2012; Meyer et al., 2012; Wang et al., 2014), which could indi- tion of the function of m A within the broader Flaviviridae family rectly regulate the HCV life cycle, for example, by inducing anti- of viruses. viral interferon-stimulated genes (ISGs). While we did not find consistent changes in ISG mRNA levels following loss of the RESULTS m A machinery during HCV infection (48 hpi), FTO depletion slightly increased the expression of IFITM1, which is known to The m A Machinery Regulates HCV Particle Production restrict HCV entry (Figure S1J) (Wilkins et al., 2013). This slight To determine whether m A regulates HCV infection, we increase occurred at both 24 and 48 hpi, although the percent- depleted the m A methyltransferases METTL3 and METTL14 age of HCV-positive cells following FTO depletion is the same as control and METTL3+14 depletion at 24 hpi (Figures 1B, (METTL3+14) by small interfering RNA (siRNA) in Huh7 liver S1J, and S1K). Therefore, the observed changes in infectious vi- hepatoma cells and infected these cells with HCV. Immunoblot analysis of cell extracts harvested at 72 hr post-infection (hpi) re- rus following depletion of the m A machinery are not solely a vealed that METTL3+14 depletion significantly increased the result of an altered antiviral response in these cells. Rather, these abundance of the HCV NS5A protein, a marker of viral replica- data suggest that m A acts directly on the HCV RNA genome to tion, relative to its level in cells treated with non-targeting control regulate HCV particle production. siRNA (Figure 1A). Conversely, depletion of the m A demethy- lase FTO decreased HCV NS5A levels relative to the control The m A-Binding YTHDF Proteins Negatively Regulate (Figure 1A). Furthermore, we found that the percentage of HCV Particle Production HCV-positive cells increased after METTL3+14 depletion and Given that the m A machinery regulates infectious HCV particle decreased after FTO depletion (Figures 1B, 1C, and S1A). This production, we next tested whether the known mediators of m A change in HCV-positive cells occurred only after 24 hpi, suggest- function, the RNA-binding YTHDF proteins, similarly regulate the ing that viral entry was unaffected by m A machinery depletion. HCV life cycle. Depletion of any of the YTHDF proteins did not in- Depletion of the m A machinery did not impair cell viability during crease HCV NS5A protein levels at 48 hpi or HCV RNA replication infection (Figure S1B). In addition, HCV infection slightly reduced of the HCV reporter (JFH1-QL/GLuc2A) over 72 hr in Huh7.5 METTL3 protein levels in total cellular extracts, while METTL14 CD81 KO cells. However, by 72 hpi, the levels of infectious Cell Host & Microbe 20, 654–665, November 9, 2016 655 6 Figure 1. The m A Machinery Regulates Infectious HCV Particle Production (A) Immunoblot analysis of extracts of HCV-infected Huh7 cells (72 hpi) treated with siRNAs. NS5A levels were quantified relative to tubulin (n = 3). *p% 0.05 by unpaired Student’s t test. (B) Percentage of HCV+ cells by immunostaining of NS5A and nuclei (DAPI) after siRNA. n = 3, withR5,000 cells counted per condition. *p% 0.05, ***p% 0.001 by two-way ANOVA with Bonferroni correction. (C) Representative fields of HCV-infected cells (NS5A , green) and nuclei (DAPI, blue) at 72 hpi from (B). (D and E) FFA of supernatants harvested from Huh7 cells 72 hpi after siRNA treatment (D). HCV RNA in supernatants harvested from Huh7 cells 72 hpi after siRNA treatment as quantified by qRT-PCR (E). Data are presented as the percentage of viral titer or RNA relative to control siRNA. ***p% 0.001 by unpaired Student’s t test. Values are the mean ± SEM of three experiments in triplicate. (F) Gaussia luciferase assay to measure HCV luciferase reporter (JFH1-QL/GLuc2A) transfected in Huh7.5 CD81 KO cells after siRNA treatment. Pol , lethal mutation in HCV NS5B polymerase. Values in (B) and (F) represent the mean ± SD (n = 3) and are representative of three independent experiments. See also Figure S1. HCV particles and HCV RNA in the supernatant were increased YTHDF Proteins Relocalize to Lipid Droplets during HCV at least 2-fold compared to control (Figures 2A–2D). Depletion Infection of YTHDF proteins did not affect cell viability, and HCV infection HCV particle assembly occurs around cytosolic lipid droplets in did not alter their expression (Figures S2A and S2B). Collectively, close association with endoplasmic reticulum (ER) membranes. these data suggest that the YTHDF proteins negatively regulate HCV RNA and proteins, including NS5A and Core (the capsid infectious HCV production without affecting overall HCV RNA protein), as well as several host RNA-binding proteins that regu- replication. late HCV infection, accumulate around lipid droplets (Ariumi We next tested whether YTHDF proteins bind to HCV RNA by et al., 2011; Chatel-Chaix et al., 2013; Miyanari et al., 2007; Pager RNA immunoprecipitation (RIP). We found that FLAG-YTHDF et al., 2013; Poenisch et al., 2015). Therefore, we analyzed the ribonucleoprotein (RNP) complexes enriched HCV RNA relative subcellular localization of YTHDF proteins after HCV infection to the input, demonstrating that these proteins bind to viral in Huh7 cells by confocal microscopy. While YTHDF proteins RNA (Figure 2E). Thus, YTHDF protein binding to HCV RNA were distributed in the cytoplasm in uninfected cells, in HCV-in- may mediate regulation of HCV particle production. This led us fected cells all three YTHDF proteins (both endogenous and to examine the subcellular localization of the YTHDF proteins overexpressed) were enriched around lipid droplets (Figures 3 during HCV infection. and S3A), in which they colocalized with the HCV Core protein. 656 Cell Host & Microbe 20, 654–665, November 9, 2016 6 Figure 2. The m A-Binding YTHDF Proteins Negatively Regulate Infectious HCV Particle Production (A) Immunoblot analysis of extracts of HCV-in- fected Huh7 cells (48 hpi) treated with indicated siRNAs. (B and C) FFA of supernatants harvested from Huh7 cells at 72 hpi after siRNA treatment (B). HCV RNA in supernatants harvested from Huh7 cells 72 hpi after siRNA treatment was quantified by qRT-PCR (C). Data were analyzed as the per- centage of titer or HCV RNA relative to cells treated with control siRNA. Values represent the mean ± SEM of three (C) or four (B) experiments done in triplicate. (D) Gaussia luciferase assay to measure HCV luciferase reporter (JFH1-QL/GLuc2A) transfected in Huh7.5 CD81 KO cells after siRNA. (E) Enrichment of HCV RNA following immuno- precipitation of FLAG-tagged YTHDF from extracts of Huh7 cells after 48 hpi. Left: captured HCV RNA was quantified by qRT-PCR as the percentage of input and graphed as fold enrichment relative to vector. Right: immunoblot analysis of immuno- precipitated extracts and input. For (D) and (E), data are representative of three experiments and show the mean ± SD (n = 3). *p % 0.05, **p % 0.01, ***p % 0.001 by unpaired Student’s t test. See also Figure S2. anti-m A antibody did not enrich HCV RNA isolated from cell supernatants to the same degree as intracellular viral RNA (Figure 4A). We next mapped the sites of the HCV RNA genome modified We did not observe this relocalization in Huh7 cells stably ex- by m A using MeRIP followed by sequencing (MeRIP-seq), as pressing a subgenomic HCV replicon that lacks the HCV struc- previously described (Dominissini et al., 2013; Meyer et al., tural genes and cannot produce viral particles (Figure S3B) 2012). We identified 19 peaks across the HCV RNA genome (Wang et al., 2003), suggesting that a fully productive HCV infec- common to both experimental replicates (Figures 4B and S6; tion is required to trigger the relocalization of the YTHDF proteins Table S1). These data present evidence that HCV, which repli- around lipid droplets. cates exclusively in the cytoplasm, is marked by m A during infection. HCV RNA Is Modified by m A As HCV replicates in the cytoplasm in association with intra- 6 6 We and others mapped m A on HIV-1 mRNA and showed that it cellular membranes, for its RNA to undergo m A modification, regulates viral gene expression (Kennedy et al., 2016; Lichinchi the m A methyltransferases must also exist in the cytoplasm. et al., 2016; Tirumuru et al., 2016). Although m A has not been Our immunoblot analysis of isolated nuclear and cytoplasmic fractions from mock or HCV-infected Huh7 cells reveals that found in RNAs from viruses that replicate in the cytoplasm, our findings (Figures 1, 2, and 3) led us to hypothesize that the METTL3, METTL14, and FTO are all present in both the nucleus HCV RNA genome is modified by m A during infection. To test and the cytoplasm, where they could interact with viral RNA this, we used an antibody that specifically recognizes m Ato (Figures S1C and S1D). This is in concordance with reports perform methyl-RNA immunoprecipitation (MeRIP) on total that have detected both METTL3 and m A-methyltransferase RNA harvested from HCV-infected cells, followed by qRT-PCR activity in cytoplasmic extracts (Chen et al., 2015; Harper to detect enriched RNAs. HCV RNA in the eluate was specifically et al., 1990; Lin et al., 2016). Therefore, these data reveal that 6 6 enriched by the anti-m A antibody, but not immunoglobulin G the m A machinery are in the cytoplasm, where they can modify (IgG), as was known m A-modified mRNA SON, but not an cytoplasmic HCV RNA. 6 6 mRNA with little m A modification, HPRT1 (Figure 4A) (Wang Because the cellular function of m A is carried out by the et al., 2014). Ultra-high-pressure liquid chromatography-tandem YTHDF proteins, which are bound to HCV RNA (Figure 2E), mass spectrometry (UPLC-MS/MS) analysis of viral RNA we hypothesized that one or more of the YTHDF proteins would captured from HCV-infected Huh7 cells using specific antisense bind to functionally relevant m A sites on the HCV RNA genome. oligonucleotides proved that HCV RNA contains m A, with a ra- We directly mapped these YTHDF-binding sites on the tio of m A/A of approximately 0.16% (Figures S4A and S4B). The viral genome using photoactivatable ribonucleoside-enhanced Cell Host & Microbe 20, 654–665, November 9, 2016 657 Figure 3. YTHDF Proteins Relocalize to Lipid Droplets during HCV Infection (A) Confocal micrographs of HCV-infected or uninfected Huh7 cells (48 hpi) immunostained with antibodies to YTHDF (green) and HCV Core (red) proteins. Lipid droplets (gray) and nuclei (blue) were labeled with BODIPY and DAPI, respectively. Zoom panels are derived from the white box in the merge panels. Scale bar, 10 mm. (B) Enrichment of YTHDF proteins around lipid droplets was quantified using ImageJ from more than ten cells analyzed and graphed in box-and-whisker plots, representing the minimum, first quartile, median, third quartile, and maximum. **p % 0.01, ***p % 0.001 by unpaired Student’s t test. See also Figure S3. crosslinking and immunoprecipitation (PAR-CLIP) in HCV-in- m A-Abrogating Mutations in the HCV E1 Genomic fected Huh7 single-cell clones stably expressing these proteins Region Increase Viral Particle Production or GFP (Figure S4C) (Hafner et al., 2010; Kennedy et al., 2016). To elucidate the functional relevance of a specific m A site on the We identified 42 different sites on the HCV RNA genome that HCV genome, we made mutations in the genome to inactivate were bound by at least one YTHDF protein, not by GFP, and con- this modification. We identified only one region of the HCV tained the characteristic T-to-C transition that derives from genome, within the viral E1 gene, that both contains m A and reverse transcription of cross-linked 4SU residues (Table S2). is bound by YTHDF proteins at sites with consensus DRA CH Surprisingly, only two high-confidence YTHDF-binding sites motifs (Tables S1 and S2). This region of the genome has been (bound by more than one YTHDF protein) overlapped with the shown to lack major RNA secondary structure (Pirakitikulr 6 6 m A peaks identified by all replicates of MeRIP-seq, and only et al., 2016) and contains a cluster of four potential m A sites 55% of the YTHDF-binding sites contained the DRA CH motif (Figure 5A). Comparative sequence analysis of the nucleotides 6 6 required for m A(Table S2). Altogether, these data build a map in these sites revealed that the first m A site is identical in 72 6 6 of m A- and YTHDF-binding sites on the HCV RNA genome. strains of genotype 2A, while the m A motif in the latter three 658 Cell Host & Microbe 20, 654–665, November 9, 2016 6 Figure 4. HCV RNA Is Modified by m A (A) MeRIP-qRT-PCR analysis of intracellular or supernatant RNA harvested from HCV-infected Huh7.5 cells (72 hpi) and immunoprecipitated with anti-m Aor IgG. Eluted RNA is quantified as a percentage of input. Values are the mean ± SD (n = 3). **p % 0.01, ***p % 0.001 by unpaired Student’s t test. (B) Map of m A-binding sites in the HCV RNA genome by MeRIP-seq (representative of two independent samples) of RNA isolated from HCV-infected Huh7 cells. Read coverage, normalized to the total number of reads mapping to the viral genome for each experiment, is in red for MeRIP-seq and in blue for input RNA-seq. Red bars indicate m A peaks identified in duplicate experiments by MeRIPPeR analysis (FDR-corrected q value < 0.05). See also Figures S4 and S6 and Tables S1 and S2. sites is conserved among 26 representative strains of HCV from WT by YTHDF2 RIP, while FLAG-YTHDF2 bound equally to different genotypes (Figure S5A). We then mutated either the A or known m A-modified mRNA SON in both conditions (Figure 5E). the C within the consensus site to a U in the four identified m A Furthermore, depletion of YTHDF1 did not increase extracellular mut mut sites in the E1 gene to construct HCV-E1 . These mutations HCV RNA produced by cells infected with E1 HCV over cells abrogate the potential for m A modification (Kane and Beemon, treated with control siRNA (Figure S5C). 1987) without altering the encoded amino acid sequence The HCV Core protein binds to the HCV RNA genome during (Figure 5A). assembly of viral particles. Core protein is known to bind to To determine the role of these m A sites in the HCV life cycle, HCV RNA around the E1 region that contains our identified mut 6 6 we electroporated wild-type (WT) and E1 HCV RNA into m A sites (Shimoike et al., 1999). To test whether m A in E1 influ- Huh7 cells and measured the production of infectious virus at ences Core binding to viral RNA, we immunoprecipitated Core mut mut 48 hpi. E1 produced nearly 3-fold more viral titer in superna- RNP complexes from cells electroporated with WT or E1 tant than WT, while the Pol RNA did not produce titer (Fig- HCV RNA. We found that mutation of the m A sites within the mut ure 5B). E1 also increased both intracellular and extracellular E1 region increases HCV RNA binding to the Core protein by titer, suggesting that these mutations increased viral particle nearly 2-fold compared to WT (Figure 5F). Altogether, these assembly (Figure S5B). To determine whether abrogation of data suggest that YTHDF proteins bind to the m A sites within the E1 m A sites affected HCV RNA replication, we then the HCV E1 region to mediate the negative regulation of infec- mut measured replication of the WT or E1 JFH1-QL/GLuc2A re- tious HCV particle production, while the Core protein binds to porter after transfection into Huh7.5 CD81 KO cells. The E1 mu- viral RNA genomes lacking m A within the E1 region for pack- tations did not alter HCV RNA replication over a time course aging into nascent viral particles. (Figure 5C) or the levels of viral Core protein (Figure 5D). Alto- 6 6 gether, these data suggest that m A within the E1 gene nega- Mapping of m A within the Viral RNA Genomes of the tively regulates infectious HCV particle production, similar to Flaviviridae Family of Viruses 6 6 our findings with depletion of the m A methyltransferases and Because we found that the HCV RNA genome contains m A, we YTHDF proteins. wanted to investigate the location of m A on the RNA genomes While the YTHDF proteins bind to multiple sites on HCV RNA, of other members of the Flaviviridae family. We performed comparison of MeRIP-seq with the PAR-CLIP data suggests that MeRIP-seq in duplicate on RNA isolated from Huh7 cells in- their binding to the HCV RNA genome is not always m A depen- fected with DENV (DENV2-NGC), YFV (17D), WNV (TX), and 6 6 dent (Figure 4B; Table S2). Therefore, to test whether the m A- ZIKV (PR2015 or DAK). Our data identified reproducible m A abrogating mutations in E1 affect binding by YTHDF proteins sites within all five viral genomes (Figures 6A–6E and S6; Table within this region, we measured FLAG-YTHDF2 binding to a re- S3). Some m A sites on these viral genomes occurred within mut porter RNA containing 100 nucleotides of WT or E1 , allowing similar genetic regions among all Flaviviridae (Figure 6F). In 6 6 us to isolate the interaction of a single m A region with a single particular, the NS3 and NS5 genes contained m A peaks, remi- YTHDF protein. Mutation of the m A sites within the E1 region niscent of the pattern on the HCV RNA genome and suggesting reduced binding of FLAG-YTHDF2 by 50% compared to the a conserved role for these sites in regulating these viral life Cell Host & Microbe 20, 654–665, November 9, 2016 659 6 Figure 5. m A-Abrogating Mutations in E1 Increase Infectious HCV Particle Production (A) Schematic of the HCV genome with the muta- tion scheme for altering A or C residues (red ar- mut 6 rows) to make the E1 virus. Consensus m A motifs (green) and inactivating mutations (red) are shown. Dashes represent nucleotides not shown. Genomic indices match the HCV JFH-1 genome (AB047639). (B) FFA of supernatants harvested from Huh7 cells mut after electroporation of WT or E1 HCV RNA (48 hr) and analyzed as the percentage of viral titer relative to WT. (C) Gaussia luciferase assay to measure levels of mut the WT, E1 , or Pol HCV luciferase reporter (JFH1-QL/GLuc2A) transfected in Huh7.5 CD81 KO cells. mut (D) Immunoblot analysis of extracts of WT, E1 , or Pol JFH1-QL/GLuc2A transfected in Huh7.5 CD81 KO cells. mut (E) Enrichment of WT or E1 reporter RNA or SON mRNA by immunoprecipitation of FLAG-YTHDF2 or vector from extracts of Huh7 cells. Captured RNA was quantified by qRT-PCR and graphed as the percentage of input. Right: immunoblot anal- ysis of anti-FLAG immunoprecipitated extracts and input. mut (F) Enrichment of WT or E1 HCV RNA by immunoprecipitation of HCV Core from extracts of Huh7 cells electroporated with the indicated viral genomes (48 hr). Lower: immunoblot analysis of anti-Core immunoprecipitated extracts and input. Data are representative of two (D and E) or three (B, C, and F) experiments and presented as the mean ± SD (n = 3). *p % 0.05, **p % 0.01, ***p% 0.001 by unpaired Student’s t test. See also Figure S5. cycles. Furthermore, similar to HCV, DENV and ZIKV (PR2015) Flaviviridae RNA genomes harbor RNA regulatory marks that contained an m A peak in the envelope gene. Therefore, these could affect their life cycles and virulence. data suggest a potentially conserved set of m A-epitranscrip- The known enzymes and RNA-binding proteins that regulate 6 6 tome sites in the Flaviviridae family that could regulate viral m A also regulate the life cycle of HCV. Depletion of the m A RNA function, virulence, and transmission. methyltransferases METTL3 and METTL14 increases the rate of HCV infection by promoting infectious viral particle production DISCUSSION without affecting viral RNA replication. Depletion of the m A de- methylase FTO, but not ALKBH5, has the opposite effect (Fig- The function of m A in regulating host and viral infection is only ure 1). These effects do not appear to be caused by dysregulated now emerging, even though nuclear-replicating viruses have induction of host ISGs after depletion of the m A machinery, been known to contain m A since the 1970s (Dimock and Stoltz- because changes in ISG expression were minimal (Figure S1). fus, 1977; Kane and Beemon, 1985; Krug et al., 1976; Lavi and Instead, we hypothesize that the m A machinery directly modu- Shatkin, 1975; Sommer et al., 1976). Recent studies have estab- lates the levels of m A on the HCV genome to regulate its func- lished a pro-viral role for m A during HIV-1 infection (Kennedy tion, and this is supported by our finding that HCV RNA contains 6 6 et al., 2016; Lichinchi et al., 2016; Tirumuru et al., 2016). In our m A. While it is known that m A functions on host mRNAs to study, in which we define function for m A and its cellular ma- regulate their stability, translation, localization, and interactions chinery in regulating the positive-strand RNA genome of the with RNA-binding proteins (Fu et al., 2014), we hypothesize 6 6 cytoplasmic virus HCV, we find that m A negatively regulates that the function of m A in HCV RNA is not due to regulation of HCV particle production. Furthermore, we find that the posi- HCV RNA stability or translation, because our studies of HCV tive-strand RNA genomes of other viruses within the Flaviviridae RNA replication using a reporter virus found no change in re- 6 6 family, including two strains of ZIKV, are modified by m Ain porter levels following depletion of the m A machinery. Rather, conserved genomic regions. Altogether, this work reveals that our data suggest that m A regulates infectious viral particle 660 Cell Host & Microbe 20, 654–665, November 9, 2016 6 Figure 6. Mapping m A in the RNA Genomes of Flaviviridae (A–E) Read coverage of Flaviviridae genomes of (A) DENV, (B) YFV, (C) ZIKV (DAK), (D) ZIKV (PR2015), and (E) WNV for one replicate of MeRIP-seq (red), and input RNA-seq (blue) from matched samples. Colored bars indicate m A peaks identified by MeRIPPeR analysis. (n = 2; FDR-corrected q value < 0.05). (F) Alignment of replicate m A sites in the genomes of DENV (red), YFV (blue), ZIKV (DAK) (orange), ZIKV (PR2015) (green), and WNV (brown). See also Figure S6 and Table S3. production through interactions of the viral RNA with host and HIV-1 infection, all three YTHDF proteins function similarly to one viral proteins. another, although they have been described to have both pro- Because the writers (METTL3+14) and an eraser (FTO) of m A and anti-HIV function (Kennedy et al., 2016; Tirumuru et al., regulated HCV particle production, it was reasonable to hypoth- 2016). During HCV infection, YTHDF regulatory function is likely esize that the m A-binding YTHDF reader proteins would have a related to their relocalization to lipid droplets, the sites of viral as- similar effect. All three YTHDF proteins bound to HCV RNA at sembly (Figure 3). Many RNA-binding proteins relocalize to lipid similar sites and their depletion increased HCV particle produc- droplets in HCV-infected cells and regulate HCV particle produc- tion, suggesting that their effect on HCV particle production was tion (Ariumi et al., 2011; Chatel-Chaix et al., 2013; Pager et al., due to binding HCV RNA (Figure 2; Table S2). Although YTHDF1 2013; Poenisch et al., 2015). Many of these proteins are known and YTHDF2 have been found to have divergent functions on to interact with YTHDF proteins, suggesting that these interac- host mRNAs, all three YTHDF proteins in our study acted simi- tions could regulate HCV particle production (Schwartz et al., larly to suppress HCV (Wang et al., 2014, 2015). Likewise, during 2014; Wang et al., 2015). Consequently, it will be important in Cell Host & Microbe 20, 654–665, November 9, 2016 661 the future to identify any YTHDF protein-protein interactions en- the known poly-U/UC pathogen-associated molecular patterns riched during HCV infection, which may point to a regulatory in the 3 UTR of the HCV genome, we did find that YTHDF2 binds network of RNA-binding proteins that affect infectious HCV par- close to this region (Table S2), so future studies can begin to ticle production. discern whether m A plays a role in HCV innate immune evasion. We found that about 50% of YTHDF protein-binding sites We found that four other Flaviviridae (DENV, YFV, ZIKV, and identified on HCV RNA using PAR-CLIP overlapped with WNV) also contained m A within their viral genomes. Because MeRIP-seq m A peaks (Figure 4). These results are similar to these viruses replicate in the cytoplasm, our data reveal that previous studies examining the overlap of YTHDF1 or YTHDF2 m A methyltransferases are functional in the cytoplasm. Similar PAR-CLIP with MeRIP-seq data, which have found about a to the results of others, we detected the m A machinery in cyto- 60% overlap (Wang et al., 2014, 2015). We hypothesize that plasmic fractions (Figure S1C) (Chen et al., 2015; Harper et al., the non-overlapping YTHDF-binding sites in HCV RNA represent 1990; Lin et al., 2016). Therefore, cellular mRNAs could also be 6 6 m A sites not detected by MeRIP-seq due to biological variation, dynamically regulated by m A modification following export technical noise, or potentially sites that might be bound by into the cytoplasm. These viruses had prominent m A peaks in YTHDF proteins in an m A-independent fashion. A report found NS5, which encodes their viral RNA-dependent RNA polymer- that YTHDF proteins bound to an in vitro transcribed, and ase, strongly suggesting the presence of a conserved RNA reg- hence non-methylated, HCV RNA genome (Rı´os-Marco et al., ulatory element here. Both DENV and ZIKV (PR2015) contained 2016). Therefore, future studies could reveal functions of the m A peaks within their envelope genes, similar to HCV, and 6 6 YTHDF proteins that are independent of m A during the HCV future studies to determine whether these m A sites also affect life cycle. production of infectious flaviviral particles will be of interest. To discern the function of an m A site on HCV RNA during While the genomic RNA structures for DENV, YFV, ZIKV, and infection, we abrogated m A modification in the E1 region of WNV have not yet been determined, these viral genomes contain mut HCV by mutation. This E1 virus produced higher viral titers specific RNA regulatory structures, especially within their UTRs. than the WT virus (Figure 5), similar to what we found with We found that two of the mosquito-transmitted viruses, DENV 6 0 METTL3+14 and YTHDF depletion and suggesting a conserved and YFV, have m A within their 3 UTRs (Figure 6F). In DENV, regulatory mechanism between both m A and the YTHDF pro- the 3 UTR has two stem loops that regulate mosquito to human teins at this site. The presence of these mutations in E1 transmission (Villordo et al., 2015). Therefore, it is possible that increased HCV RNA binding to Core protein while reducing m A patterns and functionality in the mosquito-transmitted flavi- binding to YTHDF2. This suggests that interactions of the HCV viral genomes could contribute to vector-borne transmission. 6 6 RNA with Core are regulated by m A such that viral genomes Finally, we observed clear differences in m A patterns between lacking m A in the E1 region are preferentially segregated for the Dakar and the Puerto Rican isolates of ZIKV, which represent packaging into nascent virions. Therefore, we hypothesize that the African and the Asian lineages, respectively (Haddow et al., the presence or absence of m A in E1 facilitates competition be- 2012). Because these lineages have differences in human dis- tween YTHDF protein and HCV Core binding to the viral genome, ease, with increased pathogenicity ascribed to the Asian lineage leading to the cellular retention or packaging of HCV RNA, of ZIKV (Weaver et al., 2016), the differences in regulation of respectively. these viruses by m A could contribute to these varied infection Because RNA viruses can rapidly evolve under selection pres- outcomes. 6 6 sure, the maintenance of m A sites on the HCV genome sug- In summary, we present global m A profiling of RNA viruses gests that m A must confer an evolutionary advantage to the within the Flaviviridae family. In addition, we provide evidence virus. In HCV, whose pathology is characterized by chronic pro- that an exclusively cytoplasmic RNA is modified by m A. Further- gression during infection in the liver, a slower replication rate has more, we present a role of this modification in regulating HCV been linked to persistent infection through an evasion of immune RNA function at the level of infectious viral particle production. 6 6 surveillance (Bocharov et al., 2004). Therefore, m A may boost This work sets the stage to broadly study the role of m Ain Fla- viral fitness by allowing HCV to establish slow, persistent infec- viviridae infection, transmission, and pathogenesis. This work tions. Pirakitikulr et al. (2016) identified a conserved stem loop also has the potential to uncover regulatory strategies to inhibit in the E1 coding region, just downstream of our identified m A replication by these established and emerging viral pathogens. sites, that suppresses viral particle production without affecting viral RNA replication. This raises the possibility that within the E1 EXPERIMENTAL PROCEDURES region, multiple RNA elements, including m A, play a role in segregating the RNA genome between stages of the HCV life Cell Lines cycle. Human hepatoma Huh7, Huh7.5, and Huh7.5 CD81 KO cells were grown in The function of the other m A sites on the HCV RNA genome DMEM (Mediatech) supplemented with 10% fetal bovine serum (HyClone), remains unknown. Because many of these sites do not overlap 2 5 mM HEPES, and 13 non-essential amino acids (complete [c]DMEM; with YTHDF protein-binding sites, they may directly modify Thermo Fisher Scientific). HCV-K2040 (1B) replicon cells (Wang et al., 2003) were cultured in cDMEM containing 0.2 mg/mL geneticin (Thermo Fisher Sci- HCV RNA structure or recruit alternative m A readers, such as entific). The identity of the Huh7 and Huh7.5 cell lines was verified using the HNRNPA1/B2, eIF3, or even METTL3 (Alarco´ n et al., 2015; Lin Promega GenePrint STR kit (DNA Analysis Facility, Duke University), and cells et al., 2016; Meyer et al., 2015). They may also contribute to anti- were verified as mycoplasma free by the LookOut Mycoplasma PCR detection viral innate immune evasion, because the presence of m Aon kit (Sigma). Huh7.5 CD81 KO cells were generated by CRISPR, as described RNA has been shown to reduce its activation of toll-like receptor before, with details given in the Supplemental Experimental Procedures (Hop- 3 signaling (Kariko´ et al., 2005). While we did not identify m Ain craft et al., 2016; Hopcraft and Evans, 2015). 662 Cell Host & Microbe 20, 654–665, November 9, 2016 Viral Infections and Generation of Viral Stocks Additional experimental procedures can be found in the Supplemental HCV Experimental Procedures. Infectious stocks of a cell culture-adapted strain of genotype 2A JFH1 HCV were generated and titrated by focus-forming assay (FFA), as described ACCESSION NUMBERS (Aligeti et al., 2015). HCV infections were performed at an MOI of 0.3 for 72 hr unless noted. The accession number for the raw sequencing data obtained from the MeRIP- WNV seq and PAR-CLIP and reported in this paper is GEO: GSE83438. Working stocks of WNV isolate TX 2002-HC (WNV-TX) were generated in BHK- 21 cells and titered as described (Suthar et al., 2010). WNV infections (MOI 5) SUPPLEMENTAL INFORMATION were performed in Huh7 cells for 48 hr. DENV and YFV Supplemental Information includes Supplemental Experimental Procedures, Preparation and titering of DENV2-NGC and YFV-17D stocks has been six figures, and three tables and can be found with this article online at described (Le Sommer et al., 2012; Sessions et al., 2009). DENV and YFV in- http://dx.doi.org/10.1016/j.chom.2016.09.015. fections (MOI 2) were performed for 24 hr in Huh7 cells. ZIKV AUTHOR CONTRIBUTIONS ZIKV_PR2015 (PRVABC59) stocks were prepared and titered as described (Quicke et al., 2016). ZIKV_DAK (Zika virus/A.africanus-tc/SEN/1984/41525- N.S.G., A.B.R.M., M.J.M., A.E.R., E.M.K., C.E.M., and S.M.H. designed exper- DAK) stocks were generated and titered by FFA in Vero cells (Le Sommer iments and analyzed the data. N.S.G., A.B.R.M., M.J.M., E.M.K., A.E.R., C.V., et al., 2012). ZIKV infections (MOI 2) were performed in Huh7 cells for 24 hr. J.W., J.A.G., S.E.H., K.M.Q., B.A.L., O.R.I., S.B.B., and S.M.H. performed the experiments. C.L.H., M.J.E., M.S.S., and M.A.G.-B. provided reagents. FFA for HCV Titer N.S.G., A.B.R.M., C.E.M., and S.M.H. wrote the manuscript. All authors Supernatants were collected and filtered through 0.45 mM syringe filters. Serial contributed to editing. dilutions of supernatants were used to infect naive Huh7.5 cells in triplicate wells of a 48-well plate. At either 48 or 72 hpi, cells were fixed, permeabilized, ACKNOWLEDGMENTS and immunostained with HCV NS5A antibody (1:500; gift of Charles Rice, Rockefeller University). Following binding of horseradish peroxidase (HRP)- We thank Dr. Lemon and Dr. Weeks (University of North Carolina-Chapel Hill) conjugated secondary antibody (1:500; Jackson ImmunoResearch), infected and Dr. Rice (Rockefeller University) for reagents; the Duke University Light Mi- foci were visualized with the VIP Peroxidase Substrate Kit (Vector Labora- croscopy Core Facility; the Epigenomics Core Facility at Weill Cornell; and tories) and counted at 403 magnification. Titer (in focus-forming units per members of the S.M.H. and C.E.M. labs for discussion and reading of this milliliter) was calculated as described (Gastaminza et al., 2006). To measure manuscript. This work was supported by funds from the NIH: R01AI125416 intracellular HCV titer, cells pellets were washed in PBS, resuspended in (S.M.H. and C.E.M.); 5P30AI064518 (S.M.H.); T32-CA009111 (A.E.R.); serum-free media, and subjected to five rounds of freezing and thawing in a R25EB020393, R01NS076465, and R01ES021006 (C.E.M.); R01AI089526 dry ice and ethanol bath. Lysate was cleared by centrifugation, and FFA was and R01AI101431 (M.A.G.-B.); R01DK0951250 (M.J.E.); and U19AI083019 performed as described earlier. and R56AI110516 (M.S.S.). Additional funding sources were the Duke White- head Scholarship (S.M.H.), the Ford Foundation (C.V.), the Tri-Institutional MeRIP-Seq Training Program in Computational Biology and Medicine (A.B.R.M.), STARR Poly(A)+ RNA purified from at least 75 mg total RNA (Poly(A) Purist Mag kit; (I7-A765 and I9-A9-071; C.E.M.), the Irma T. Hirschl and Monique Weill- Thermo Fisher Scientific) extracted from HCV-, DENV-, YFV-, WNV-, ZIKV Caulier Charitable Trusts, the Bert L. and N. Kuggie Vallee Foundation, World- (DAK)-, and ZIKV (PR2015)-infected samples was fragmented using the Am- Quant, the Pershing Square Sohn Cancer Research Alliance, NASA bion RNA fragmentation reagent and purified by ethanol precipitation. Frag- (NNX14AH50G and 15-15Omni2-0063), the Bill and Melinda Gates Foundation mented RNA was heated to 75 C for 5 min, placed on ice for 3 min, and (OPP1151054), and the Alfred P. Sloan Foundation (G-2015-13964), the U-TX then incubated with anti-m A antibody (5 mg; Synaptic Systems, #202111) STARs Award (M.A.G.-B.), UTMB (M.A.G.-B. and S.S.B.), Pew Charitable conjugated to Protein G Dynabeads (50 mL; Thermo Fisher Scientific) in MeRIP Trusts (USPHS-AI07647 and ACS-RSG-12-176-01-MPC; M.J.E.), and the buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 0.1% NP-40) Burroughs Wellcome Fund. overnight at 4 C. Beads were then washed 53 with MeRIP buffer, and bound RNA was eluted in MeRIP buffer containing 6.7 mM m A sodium salt (Sigma). Received: June 17, 2016 Eluted RNA was purified with the Quick-RNA miniprep kit (Zymo Research) Revised: August 31, 2016 and concentrated by ethanol precipitation. Sequencing libraries were pre- Accepted: September 28, 2016 pared from this RNA, as well as input RNA, using the TruSeq RNA sequencing Published: October 20, 2016 (RNA-seq) kit (Illumina). 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N6 -Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

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Article N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection Graphical Abstract Authors Nandan S. Gokhale, Alexa B.R. McIntyre, Michael J. McFadden, ..., Shelton S. Bradrick, Christopher E. Mason, Stacy M. Horner Correspondence [email protected] (C.E.M.), [email protected] (S.M.H.) In Brief N6-methyladenosine (m A) post- transcriptionally regulates RNA function. Gokhale et al. demonstrate that the RNA genomes of HCV, ZIKV, DENV, YFV, and WNV are modified by m A. Depletion of cellular machinery that regulates m Aor introduction of m A-abrogating mutations within HCV RNA increase viral particle production, suggesting that m A negatively regulates HCV. Highlights Accession Numbers d The RNA genomes of HCV, ZIKV, DENV, YFV, and WNV GSE83438 contain m A modification d The cellular m A machinery regulates HCV infectious particle production d YTHDF proteins reduce HCV particle production and localize at viral assembly sites d m A-abrogating mutations in HCV E1 increase infectious particle production Gokhale et al., 2016, Cell Host & Microbe 20, 654–665 November 9, 2016 ª 2016 The Authors. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.chom.2016.09.015 Cell Host & Microbe Article N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection 1 3,12 1 1 1 Nandan S. Gokhale, Alexa B.R. McIntyre, Michael J. McFadden, Allison E. Roder, Edward M. Kennedy, 3 4 5,6 1 1 7 Jorge A. Gandara, Sharon E. Hopcraft, Kendra M. Quicke, Christine Vazquez, Jason Willer, Olga R. Ilkayeva, 2 2 8,11 4 5,6 Brittany A. Law, Christopher L. Holley, Mariano A. Garcia-Blanco, Matthew J. Evans, Mehul S. Suthar, 8 3,9,10,14, 1,2,13,15, Shelton S. Bradrick, Christopher E. Mason, * and Stacy M. Horner * Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10021, USA Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, GA 30329, USA Duke Molecular Physiology Institute, Duke University, Durham NC 27701, USA Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY 10021, USA The Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY 10021, USA Programme in Emerging Infectious Disease, Duke-NUS Medical School, Singapore 169857, Singapore Tri-Institutional Program in Computational Biology and Medicine, New York City, NY 10065, USA Lead Contact Twitter: @mason_lab Twitter: @thehornerlab *Correspondence: [email protected] (C.E.M.), [email protected] (S.M.H.) http://dx.doi.org/10.1016/j.chom.2016.09.015 SUMMARY tions, N6-methyladenosine (m A) is the most abundant internal modification of eukaryotic mRNAs, contributing to RNA struc- The RNA modification N6-methyladenosine (m A) ture, localization, and function (Fu et al., 2014; Meyer and Jaf- frey, 2014). m A regulates many biological processes, including post-transcriptionally regulates RNA function. The stress responses, fertility, stem cell differentiation, circadian cellular machinery that controls m A includes methyl- rhythms, microRNA (miRNA) biogenesis, and cancer (Li and transferases and demethylases that add or remove Mason, 2014; Saletore et al., 2012; Yue et al., 2015; Zhou this modification, as well as m A-binding YTHDF pro- et al., 2015). However, little is known about its effects on micro- teins that promote the translation or degradation of bial infection. m A has long been known to be present in the 6 6 m A-modified mRNA.We demonstratethat m A mod- RNA transcripts of viruses with nuclear replication, such as influ- ulates infection by hepatitis C virus (HCV). Depletion enza A virus, simian virus 40, Rous sarcoma virus, avian sar- 6 6 of m A methyltransferases or an m A demethylase, coma virus, and adenovirus (Dimock and Stoltzfus, 1977; respectively, increases or decreases infectious HCV Kane and Beemon, 1985; Krug et al., 1976; Lavi and Shatkin, particle production. During HCV infection, YTHDF 1975; Sommer et al., 1976). More recently, we and others proteins relocalize to lipid droplets, sites of viral as- have shown that m A serves as a positive regulator of HIV-1, sembly, and their depletion increases infectious viral a retrovirus with a nuclear replication step (Kennedy et al., 2016; Lichinchi et al., 2016; Tirumuru et al., 2016). However, a particles. We further mapped m A sites across the role for m A in regulating the life cycle of viruses that replicate HCV genome and determined that inactivating m A exclusively in the cytoplasm, such as viruses within the Flavivir- in one viral genomic region increases viral titer without idae family, has been unexplored. Flaviviridae, including Zika affecting RNA replication. Additional mapping of m A virus (ZIKV), dengue virus (DENV), West Nile virus (WNV), yellow on the RNA genomes of other Flaviviridae, including fever virus (YFV), and hepatitis C virus (HCV), represent both dengue, Zika, yellow fever, and West Nile virus, iden- established and emerging pathogens. They contain a positive- tifies conserved regions modified by m A. Altogether, sense, single-stranded RNA genome that encodes a viral this work identifies m A as a conserved regulatory polyprotein and use similar replication strategies. RNA-based mark across Flaviviridae genomes. regulation of these viral genomes plays a fundamental role in their infection, such as the liver-specific miRNA miR-122 for HCV replication, RNA structural elements for HCV and DENV 0 0 INTRODUCTION replication, and 2 -O methylation of the 5 cap of WNV RNA for immune evasion and WNV replication (Bidet and Garcia- The chemical modification of RNA is an important post-tran- Blanco, 2014; Hyde et al., 2014; Jopling et al., 2005; Mauger scriptional regulator of RNA. Of the many known RNA modifica- et al., 2015; Pirakitikulr et al., 2016). 654 Cell Host & Microbe 20, 654–665, November 9, 2016 ª 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 6 The cellular machinery that regulates m A includes proteins and FTO were unaffected (Figures S1C and S1D). Thus, the 6 6 that act as writers, erasers, and readers of m A. The addition m A methyltransferases negatively regulate HCV infection, while 6 m 6 of m A on mRNA, which occurs at the consensus motif DRA CH the m A demethylase positively regulates HCV infection. (where D = G/A/U, R = G > A, and H = U/C/A), is mediated by a We next defined the stage of the HCV life cycle regulated by methyltransferase complex containing the methyltransferase- the m A machinery. Depletion of METTL3+14 significantly like (METTL) enzymes METTL3 and METTL14 and the cofactors increased the production of infectious virus and viral RNA in Wilms tumor 1-associated protein (WTAP) and KIAA1429 (Fu the supernatant compared to control siRNA at 72 hpi (Figures et al., 2014; Liu et al., 2014; Meyer and Jaffrey, 2014; Schwartz 1D and 1E). Conversely, depletion of FTO decreased infectious et al., 2014; Yue et al., 2015). The removal of m A from mRNA virus and HCV RNA in the supernatant (Figures 1D and 1E) is catalyzed by the demethylases fat mass and obesity-associ- without altering the viral-specific infectivity (Figure S1E). Deple- ated protein (FTO) or a-ketoglutarate-dependent dioxygenase tion of ALKBH5 did not affect viral titer or protein levels, indi- AlkB homolog 5 (ALKBH5) (Jia et al., 2011; Zheng et al., 2013). cating that this demethylase does not influence the HCV life The cytoplasmic YTH-domain family 1 (YTHDF1), YTHDF2, and cycle (Figure S1F). We next tested whether the altered HCV titer 6 6 YTHDF3 proteins bind to m A through their C-terminal YTH after m A machinery depletion was due to altered viral RNA repli- domain. Functionally, YTHDF1 promotes the translation of cation. In these experiments, we used Huh7.5 CD81 knockout 6 6 m A-modified mRNA, while YTHDF2 targets m A-modified (KO) cells, in which essential HCV entry factor CD81 (Zhang mRNAs for degradation (Wang et al., 2014, 2015). The function et al., 2004) was deleted by clustered regularly interspaced short of YTHDF3 is still unknown. The discovery of these proteins palindromic repeats/Cas9 (CRISPR/Cas9), resulting in cells and the development of high-throughput m A-mapping tech- permissive for HCV RNA replication and viral particle production niques have led to many insights into the function of m A(Dom- following viral RNA transfection that are unable to support sub- inissini et al., 2012; Fu et al., 2014; Linder et al., 2015; Meyer sequent rounds of viral infection (Figures S1G–S1I). In these et al., 2012). Nonetheless, many aspects of the regulation of spe- cells, we depleted METTL3+14 or FTO by siRNA, transfected cific mRNAs by m A remain unexplored. the cells with in vitro transcribed RNA of the HCV reporter virus Here, we define a role for m A in regulating the life cycle JFH1-QL/GLuc2A, and measured HCV RNA replication by as- of HCV. We demonstrate that the m A methyltransferases saying for secreted Gaussia luciferase (Yamane et al., 2014). negatively regulate the production of infectious HCV particles Depletion of METTL3+14 or FTO had no effect on Gaussia lucif- and that the m A-binding YTHDF proteins all relocalize to sites erase levels compared to control over the time course, while our of HCV particle production and suppress this stage of viral negative control RNA containing a point mutation in the viral infection. We map m A across the HCV RNA genome and RNA-dependent RNA polymerase (Pol ) did not replicate (Fig- 6 6 show that preventing m A at one of these regions enhances ure 1F). These data indicate that m A dynamics do not regulate viral titer by increasing the interaction of the HCV RNA with HCV translation or RNA replication but do regulate the produc- the HCV Core protein. Finally, we describe viral RNA m A-epi- tion or release of infectious viral particles. transcriptomic maps for several other Flaviviridae, including Changes in expression of the m A machinery have been ZIKV, DENV, WNV, and YFV. Altogether, our data reveal that shown to affect cellular gene expression (Dominissini et al., m A regulates HCV infection and set the stage for the explora- 2012; Meyer et al., 2012; Wang et al., 2014), which could indi- tion of the function of m A within the broader Flaviviridae family rectly regulate the HCV life cycle, for example, by inducing anti- of viruses. viral interferon-stimulated genes (ISGs). While we did not find consistent changes in ISG mRNA levels following loss of the RESULTS m A machinery during HCV infection (48 hpi), FTO depletion slightly increased the expression of IFITM1, which is known to The m A Machinery Regulates HCV Particle Production restrict HCV entry (Figure S1J) (Wilkins et al., 2013). This slight To determine whether m A regulates HCV infection, we increase occurred at both 24 and 48 hpi, although the percent- depleted the m A methyltransferases METTL3 and METTL14 age of HCV-positive cells following FTO depletion is the same as control and METTL3+14 depletion at 24 hpi (Figures 1B, (METTL3+14) by small interfering RNA (siRNA) in Huh7 liver S1J, and S1K). Therefore, the observed changes in infectious vi- hepatoma cells and infected these cells with HCV. Immunoblot analysis of cell extracts harvested at 72 hr post-infection (hpi) re- rus following depletion of the m A machinery are not solely a vealed that METTL3+14 depletion significantly increased the result of an altered antiviral response in these cells. Rather, these abundance of the HCV NS5A protein, a marker of viral replica- data suggest that m A acts directly on the HCV RNA genome to tion, relative to its level in cells treated with non-targeting control regulate HCV particle production. siRNA (Figure 1A). Conversely, depletion of the m A demethy- lase FTO decreased HCV NS5A levels relative to the control The m A-Binding YTHDF Proteins Negatively Regulate (Figure 1A). Furthermore, we found that the percentage of HCV Particle Production HCV-positive cells increased after METTL3+14 depletion and Given that the m A machinery regulates infectious HCV particle decreased after FTO depletion (Figures 1B, 1C, and S1A). This production, we next tested whether the known mediators of m A change in HCV-positive cells occurred only after 24 hpi, suggest- function, the RNA-binding YTHDF proteins, similarly regulate the ing that viral entry was unaffected by m A machinery depletion. HCV life cycle. Depletion of any of the YTHDF proteins did not in- Depletion of the m A machinery did not impair cell viability during crease HCV NS5A protein levels at 48 hpi or HCV RNA replication infection (Figure S1B). In addition, HCV infection slightly reduced of the HCV reporter (JFH1-QL/GLuc2A) over 72 hr in Huh7.5 METTL3 protein levels in total cellular extracts, while METTL14 CD81 KO cells. However, by 72 hpi, the levels of infectious Cell Host & Microbe 20, 654–665, November 9, 2016 655 6 Figure 1. The m A Machinery Regulates Infectious HCV Particle Production (A) Immunoblot analysis of extracts of HCV-infected Huh7 cells (72 hpi) treated with siRNAs. NS5A levels were quantified relative to tubulin (n = 3). *p% 0.05 by unpaired Student’s t test. (B) Percentage of HCV+ cells by immunostaining of NS5A and nuclei (DAPI) after siRNA. n = 3, withR5,000 cells counted per condition. *p% 0.05, ***p% 0.001 by two-way ANOVA with Bonferroni correction. (C) Representative fields of HCV-infected cells (NS5A , green) and nuclei (DAPI, blue) at 72 hpi from (B). (D and E) FFA of supernatants harvested from Huh7 cells 72 hpi after siRNA treatment (D). HCV RNA in supernatants harvested from Huh7 cells 72 hpi after siRNA treatment as quantified by qRT-PCR (E). Data are presented as the percentage of viral titer or RNA relative to control siRNA. ***p% 0.001 by unpaired Student’s t test. Values are the mean ± SEM of three experiments in triplicate. (F) Gaussia luciferase assay to measure HCV luciferase reporter (JFH1-QL/GLuc2A) transfected in Huh7.5 CD81 KO cells after siRNA treatment. Pol , lethal mutation in HCV NS5B polymerase. Values in (B) and (F) represent the mean ± SD (n = 3) and are representative of three independent experiments. See also Figure S1. HCV particles and HCV RNA in the supernatant were increased YTHDF Proteins Relocalize to Lipid Droplets during HCV at least 2-fold compared to control (Figures 2A–2D). Depletion Infection of YTHDF proteins did not affect cell viability, and HCV infection HCV particle assembly occurs around cytosolic lipid droplets in did not alter their expression (Figures S2A and S2B). Collectively, close association with endoplasmic reticulum (ER) membranes. these data suggest that the YTHDF proteins negatively regulate HCV RNA and proteins, including NS5A and Core (the capsid infectious HCV production without affecting overall HCV RNA protein), as well as several host RNA-binding proteins that regu- replication. late HCV infection, accumulate around lipid droplets (Ariumi We next tested whether YTHDF proteins bind to HCV RNA by et al., 2011; Chatel-Chaix et al., 2013; Miyanari et al., 2007; Pager RNA immunoprecipitation (RIP). We found that FLAG-YTHDF et al., 2013; Poenisch et al., 2015). Therefore, we analyzed the ribonucleoprotein (RNP) complexes enriched HCV RNA relative subcellular localization of YTHDF proteins after HCV infection to the input, demonstrating that these proteins bind to viral in Huh7 cells by confocal microscopy. While YTHDF proteins RNA (Figure 2E). Thus, YTHDF protein binding to HCV RNA were distributed in the cytoplasm in uninfected cells, in HCV-in- may mediate regulation of HCV particle production. This led us fected cells all three YTHDF proteins (both endogenous and to examine the subcellular localization of the YTHDF proteins overexpressed) were enriched around lipid droplets (Figures 3 during HCV infection. and S3A), in which they colocalized with the HCV Core protein. 656 Cell Host & Microbe 20, 654–665, November 9, 2016 6 Figure 2. The m A-Binding YTHDF Proteins Negatively Regulate Infectious HCV Particle Production (A) Immunoblot analysis of extracts of HCV-in- fected Huh7 cells (48 hpi) treated with indicated siRNAs. (B and C) FFA of supernatants harvested from Huh7 cells at 72 hpi after siRNA treatment (B). HCV RNA in supernatants harvested from Huh7 cells 72 hpi after siRNA treatment was quantified by qRT-PCR (C). Data were analyzed as the per- centage of titer or HCV RNA relative to cells treated with control siRNA. Values represent the mean ± SEM of three (C) or four (B) experiments done in triplicate. (D) Gaussia luciferase assay to measure HCV luciferase reporter (JFH1-QL/GLuc2A) transfected in Huh7.5 CD81 KO cells after siRNA. (E) Enrichment of HCV RNA following immuno- precipitation of FLAG-tagged YTHDF from extracts of Huh7 cells after 48 hpi. Left: captured HCV RNA was quantified by qRT-PCR as the percentage of input and graphed as fold enrichment relative to vector. Right: immunoblot analysis of immuno- precipitated extracts and input. For (D) and (E), data are representative of three experiments and show the mean ± SD (n = 3). *p % 0.05, **p % 0.01, ***p % 0.001 by unpaired Student’s t test. See also Figure S2. anti-m A antibody did not enrich HCV RNA isolated from cell supernatants to the same degree as intracellular viral RNA (Figure 4A). We next mapped the sites of the HCV RNA genome modified We did not observe this relocalization in Huh7 cells stably ex- by m A using MeRIP followed by sequencing (MeRIP-seq), as pressing a subgenomic HCV replicon that lacks the HCV struc- previously described (Dominissini et al., 2013; Meyer et al., tural genes and cannot produce viral particles (Figure S3B) 2012). We identified 19 peaks across the HCV RNA genome (Wang et al., 2003), suggesting that a fully productive HCV infec- common to both experimental replicates (Figures 4B and S6; tion is required to trigger the relocalization of the YTHDF proteins Table S1). These data present evidence that HCV, which repli- around lipid droplets. cates exclusively in the cytoplasm, is marked by m A during infection. HCV RNA Is Modified by m A As HCV replicates in the cytoplasm in association with intra- 6 6 We and others mapped m A on HIV-1 mRNA and showed that it cellular membranes, for its RNA to undergo m A modification, regulates viral gene expression (Kennedy et al., 2016; Lichinchi the m A methyltransferases must also exist in the cytoplasm. et al., 2016; Tirumuru et al., 2016). Although m A has not been Our immunoblot analysis of isolated nuclear and cytoplasmic fractions from mock or HCV-infected Huh7 cells reveals that found in RNAs from viruses that replicate in the cytoplasm, our findings (Figures 1, 2, and 3) led us to hypothesize that the METTL3, METTL14, and FTO are all present in both the nucleus HCV RNA genome is modified by m A during infection. To test and the cytoplasm, where they could interact with viral RNA this, we used an antibody that specifically recognizes m Ato (Figures S1C and S1D). This is in concordance with reports perform methyl-RNA immunoprecipitation (MeRIP) on total that have detected both METTL3 and m A-methyltransferase RNA harvested from HCV-infected cells, followed by qRT-PCR activity in cytoplasmic extracts (Chen et al., 2015; Harper to detect enriched RNAs. HCV RNA in the eluate was specifically et al., 1990; Lin et al., 2016). Therefore, these data reveal that 6 6 enriched by the anti-m A antibody, but not immunoglobulin G the m A machinery are in the cytoplasm, where they can modify (IgG), as was known m A-modified mRNA SON, but not an cytoplasmic HCV RNA. 6 6 mRNA with little m A modification, HPRT1 (Figure 4A) (Wang Because the cellular function of m A is carried out by the et al., 2014). Ultra-high-pressure liquid chromatography-tandem YTHDF proteins, which are bound to HCV RNA (Figure 2E), mass spectrometry (UPLC-MS/MS) analysis of viral RNA we hypothesized that one or more of the YTHDF proteins would captured from HCV-infected Huh7 cells using specific antisense bind to functionally relevant m A sites on the HCV RNA genome. oligonucleotides proved that HCV RNA contains m A, with a ra- We directly mapped these YTHDF-binding sites on the tio of m A/A of approximately 0.16% (Figures S4A and S4B). The viral genome using photoactivatable ribonucleoside-enhanced Cell Host & Microbe 20, 654–665, November 9, 2016 657 Figure 3. YTHDF Proteins Relocalize to Lipid Droplets during HCV Infection (A) Confocal micrographs of HCV-infected or uninfected Huh7 cells (48 hpi) immunostained with antibodies to YTHDF (green) and HCV Core (red) proteins. Lipid droplets (gray) and nuclei (blue) were labeled with BODIPY and DAPI, respectively. Zoom panels are derived from the white box in the merge panels. Scale bar, 10 mm. (B) Enrichment of YTHDF proteins around lipid droplets was quantified using ImageJ from more than ten cells analyzed and graphed in box-and-whisker plots, representing the minimum, first quartile, median, third quartile, and maximum. **p % 0.01, ***p % 0.001 by unpaired Student’s t test. See also Figure S3. crosslinking and immunoprecipitation (PAR-CLIP) in HCV-in- m A-Abrogating Mutations in the HCV E1 Genomic fected Huh7 single-cell clones stably expressing these proteins Region Increase Viral Particle Production or GFP (Figure S4C) (Hafner et al., 2010; Kennedy et al., 2016). To elucidate the functional relevance of a specific m A site on the We identified 42 different sites on the HCV RNA genome that HCV genome, we made mutations in the genome to inactivate were bound by at least one YTHDF protein, not by GFP, and con- this modification. We identified only one region of the HCV tained the characteristic T-to-C transition that derives from genome, within the viral E1 gene, that both contains m A and reverse transcription of cross-linked 4SU residues (Table S2). is bound by YTHDF proteins at sites with consensus DRA CH Surprisingly, only two high-confidence YTHDF-binding sites motifs (Tables S1 and S2). This region of the genome has been (bound by more than one YTHDF protein) overlapped with the shown to lack major RNA secondary structure (Pirakitikulr 6 6 m A peaks identified by all replicates of MeRIP-seq, and only et al., 2016) and contains a cluster of four potential m A sites 55% of the YTHDF-binding sites contained the DRA CH motif (Figure 5A). Comparative sequence analysis of the nucleotides 6 6 required for m A(Table S2). Altogether, these data build a map in these sites revealed that the first m A site is identical in 72 6 6 of m A- and YTHDF-binding sites on the HCV RNA genome. strains of genotype 2A, while the m A motif in the latter three 658 Cell Host & Microbe 20, 654–665, November 9, 2016 6 Figure 4. HCV RNA Is Modified by m A (A) MeRIP-qRT-PCR analysis of intracellular or supernatant RNA harvested from HCV-infected Huh7.5 cells (72 hpi) and immunoprecipitated with anti-m Aor IgG. Eluted RNA is quantified as a percentage of input. Values are the mean ± SD (n = 3). **p % 0.01, ***p % 0.001 by unpaired Student’s t test. (B) Map of m A-binding sites in the HCV RNA genome by MeRIP-seq (representative of two independent samples) of RNA isolated from HCV-infected Huh7 cells. Read coverage, normalized to the total number of reads mapping to the viral genome for each experiment, is in red for MeRIP-seq and in blue for input RNA-seq. Red bars indicate m A peaks identified in duplicate experiments by MeRIPPeR analysis (FDR-corrected q value < 0.05). See also Figures S4 and S6 and Tables S1 and S2. sites is conserved among 26 representative strains of HCV from WT by YTHDF2 RIP, while FLAG-YTHDF2 bound equally to different genotypes (Figure S5A). We then mutated either the A or known m A-modified mRNA SON in both conditions (Figure 5E). the C within the consensus site to a U in the four identified m A Furthermore, depletion of YTHDF1 did not increase extracellular mut mut sites in the E1 gene to construct HCV-E1 . These mutations HCV RNA produced by cells infected with E1 HCV over cells abrogate the potential for m A modification (Kane and Beemon, treated with control siRNA (Figure S5C). 1987) without altering the encoded amino acid sequence The HCV Core protein binds to the HCV RNA genome during (Figure 5A). assembly of viral particles. Core protein is known to bind to To determine the role of these m A sites in the HCV life cycle, HCV RNA around the E1 region that contains our identified mut 6 6 we electroporated wild-type (WT) and E1 HCV RNA into m A sites (Shimoike et al., 1999). To test whether m A in E1 influ- Huh7 cells and measured the production of infectious virus at ences Core binding to viral RNA, we immunoprecipitated Core mut mut 48 hpi. E1 produced nearly 3-fold more viral titer in superna- RNP complexes from cells electroporated with WT or E1 tant than WT, while the Pol RNA did not produce titer (Fig- HCV RNA. We found that mutation of the m A sites within the mut ure 5B). E1 also increased both intracellular and extracellular E1 region increases HCV RNA binding to the Core protein by titer, suggesting that these mutations increased viral particle nearly 2-fold compared to WT (Figure 5F). Altogether, these assembly (Figure S5B). To determine whether abrogation of data suggest that YTHDF proteins bind to the m A sites within the E1 m A sites affected HCV RNA replication, we then the HCV E1 region to mediate the negative regulation of infec- mut measured replication of the WT or E1 JFH1-QL/GLuc2A re- tious HCV particle production, while the Core protein binds to porter after transfection into Huh7.5 CD81 KO cells. The E1 mu- viral RNA genomes lacking m A within the E1 region for pack- tations did not alter HCV RNA replication over a time course aging into nascent viral particles. (Figure 5C) or the levels of viral Core protein (Figure 5D). Alto- 6 6 gether, these data suggest that m A within the E1 gene nega- Mapping of m A within the Viral RNA Genomes of the tively regulates infectious HCV particle production, similar to Flaviviridae Family of Viruses 6 6 our findings with depletion of the m A methyltransferases and Because we found that the HCV RNA genome contains m A, we YTHDF proteins. wanted to investigate the location of m A on the RNA genomes While the YTHDF proteins bind to multiple sites on HCV RNA, of other members of the Flaviviridae family. We performed comparison of MeRIP-seq with the PAR-CLIP data suggests that MeRIP-seq in duplicate on RNA isolated from Huh7 cells in- their binding to the HCV RNA genome is not always m A depen- fected with DENV (DENV2-NGC), YFV (17D), WNV (TX), and 6 6 dent (Figure 4B; Table S2). Therefore, to test whether the m A- ZIKV (PR2015 or DAK). Our data identified reproducible m A abrogating mutations in E1 affect binding by YTHDF proteins sites within all five viral genomes (Figures 6A–6E and S6; Table within this region, we measured FLAG-YTHDF2 binding to a re- S3). Some m A sites on these viral genomes occurred within mut porter RNA containing 100 nucleotides of WT or E1 , allowing similar genetic regions among all Flaviviridae (Figure 6F). In 6 6 us to isolate the interaction of a single m A region with a single particular, the NS3 and NS5 genes contained m A peaks, remi- YTHDF protein. Mutation of the m A sites within the E1 region niscent of the pattern on the HCV RNA genome and suggesting reduced binding of FLAG-YTHDF2 by 50% compared to the a conserved role for these sites in regulating these viral life Cell Host & Microbe 20, 654–665, November 9, 2016 659 6 Figure 5. m A-Abrogating Mutations in E1 Increase Infectious HCV Particle Production (A) Schematic of the HCV genome with the muta- tion scheme for altering A or C residues (red ar- mut 6 rows) to make the E1 virus. Consensus m A motifs (green) and inactivating mutations (red) are shown. Dashes represent nucleotides not shown. Genomic indices match the HCV JFH-1 genome (AB047639). (B) FFA of supernatants harvested from Huh7 cells mut after electroporation of WT or E1 HCV RNA (48 hr) and analyzed as the percentage of viral titer relative to WT. (C) Gaussia luciferase assay to measure levels of mut the WT, E1 , or Pol HCV luciferase reporter (JFH1-QL/GLuc2A) transfected in Huh7.5 CD81 KO cells. mut (D) Immunoblot analysis of extracts of WT, E1 , or Pol JFH1-QL/GLuc2A transfected in Huh7.5 CD81 KO cells. mut (E) Enrichment of WT or E1 reporter RNA or SON mRNA by immunoprecipitation of FLAG-YTHDF2 or vector from extracts of Huh7 cells. Captured RNA was quantified by qRT-PCR and graphed as the percentage of input. Right: immunoblot anal- ysis of anti-FLAG immunoprecipitated extracts and input. mut (F) Enrichment of WT or E1 HCV RNA by immunoprecipitation of HCV Core from extracts of Huh7 cells electroporated with the indicated viral genomes (48 hr). Lower: immunoblot analysis of anti-Core immunoprecipitated extracts and input. Data are representative of two (D and E) or three (B, C, and F) experiments and presented as the mean ± SD (n = 3). *p % 0.05, **p % 0.01, ***p% 0.001 by unpaired Student’s t test. See also Figure S5. cycles. Furthermore, similar to HCV, DENV and ZIKV (PR2015) Flaviviridae RNA genomes harbor RNA regulatory marks that contained an m A peak in the envelope gene. Therefore, these could affect their life cycles and virulence. data suggest a potentially conserved set of m A-epitranscrip- The known enzymes and RNA-binding proteins that regulate 6 6 tome sites in the Flaviviridae family that could regulate viral m A also regulate the life cycle of HCV. Depletion of the m A RNA function, virulence, and transmission. methyltransferases METTL3 and METTL14 increases the rate of HCV infection by promoting infectious viral particle production DISCUSSION without affecting viral RNA replication. Depletion of the m A de- methylase FTO, but not ALKBH5, has the opposite effect (Fig- The function of m A in regulating host and viral infection is only ure 1). These effects do not appear to be caused by dysregulated now emerging, even though nuclear-replicating viruses have induction of host ISGs after depletion of the m A machinery, been known to contain m A since the 1970s (Dimock and Stoltz- because changes in ISG expression were minimal (Figure S1). fus, 1977; Kane and Beemon, 1985; Krug et al., 1976; Lavi and Instead, we hypothesize that the m A machinery directly modu- Shatkin, 1975; Sommer et al., 1976). Recent studies have estab- lates the levels of m A on the HCV genome to regulate its func- lished a pro-viral role for m A during HIV-1 infection (Kennedy tion, and this is supported by our finding that HCV RNA contains 6 6 et al., 2016; Lichinchi et al., 2016; Tirumuru et al., 2016). In our m A. While it is known that m A functions on host mRNAs to study, in which we define function for m A and its cellular ma- regulate their stability, translation, localization, and interactions chinery in regulating the positive-strand RNA genome of the with RNA-binding proteins (Fu et al., 2014), we hypothesize 6 6 cytoplasmic virus HCV, we find that m A negatively regulates that the function of m A in HCV RNA is not due to regulation of HCV particle production. Furthermore, we find that the posi- HCV RNA stability or translation, because our studies of HCV tive-strand RNA genomes of other viruses within the Flaviviridae RNA replication using a reporter virus found no change in re- 6 6 family, including two strains of ZIKV, are modified by m Ain porter levels following depletion of the m A machinery. Rather, conserved genomic regions. Altogether, this work reveals that our data suggest that m A regulates infectious viral particle 660 Cell Host & Microbe 20, 654–665, November 9, 2016 6 Figure 6. Mapping m A in the RNA Genomes of Flaviviridae (A–E) Read coverage of Flaviviridae genomes of (A) DENV, (B) YFV, (C) ZIKV (DAK), (D) ZIKV (PR2015), and (E) WNV for one replicate of MeRIP-seq (red), and input RNA-seq (blue) from matched samples. Colored bars indicate m A peaks identified by MeRIPPeR analysis. (n = 2; FDR-corrected q value < 0.05). (F) Alignment of replicate m A sites in the genomes of DENV (red), YFV (blue), ZIKV (DAK) (orange), ZIKV (PR2015) (green), and WNV (brown). See also Figure S6 and Table S3. production through interactions of the viral RNA with host and HIV-1 infection, all three YTHDF proteins function similarly to one viral proteins. another, although they have been described to have both pro- Because the writers (METTL3+14) and an eraser (FTO) of m A and anti-HIV function (Kennedy et al., 2016; Tirumuru et al., regulated HCV particle production, it was reasonable to hypoth- 2016). During HCV infection, YTHDF regulatory function is likely esize that the m A-binding YTHDF reader proteins would have a related to their relocalization to lipid droplets, the sites of viral as- similar effect. All three YTHDF proteins bound to HCV RNA at sembly (Figure 3). Many RNA-binding proteins relocalize to lipid similar sites and their depletion increased HCV particle produc- droplets in HCV-infected cells and regulate HCV particle produc- tion, suggesting that their effect on HCV particle production was tion (Ariumi et al., 2011; Chatel-Chaix et al., 2013; Pager et al., due to binding HCV RNA (Figure 2; Table S2). Although YTHDF1 2013; Poenisch et al., 2015). Many of these proteins are known and YTHDF2 have been found to have divergent functions on to interact with YTHDF proteins, suggesting that these interac- host mRNAs, all three YTHDF proteins in our study acted simi- tions could regulate HCV particle production (Schwartz et al., larly to suppress HCV (Wang et al., 2014, 2015). Likewise, during 2014; Wang et al., 2015). Consequently, it will be important in Cell Host & Microbe 20, 654–665, November 9, 2016 661 the future to identify any YTHDF protein-protein interactions en- the known poly-U/UC pathogen-associated molecular patterns riched during HCV infection, which may point to a regulatory in the 3 UTR of the HCV genome, we did find that YTHDF2 binds network of RNA-binding proteins that affect infectious HCV par- close to this region (Table S2), so future studies can begin to ticle production. discern whether m A plays a role in HCV innate immune evasion. We found that about 50% of YTHDF protein-binding sites We found that four other Flaviviridae (DENV, YFV, ZIKV, and identified on HCV RNA using PAR-CLIP overlapped with WNV) also contained m A within their viral genomes. Because MeRIP-seq m A peaks (Figure 4). These results are similar to these viruses replicate in the cytoplasm, our data reveal that previous studies examining the overlap of YTHDF1 or YTHDF2 m A methyltransferases are functional in the cytoplasm. Similar PAR-CLIP with MeRIP-seq data, which have found about a to the results of others, we detected the m A machinery in cyto- 60% overlap (Wang et al., 2014, 2015). We hypothesize that plasmic fractions (Figure S1C) (Chen et al., 2015; Harper et al., the non-overlapping YTHDF-binding sites in HCV RNA represent 1990; Lin et al., 2016). Therefore, cellular mRNAs could also be 6 6 m A sites not detected by MeRIP-seq due to biological variation, dynamically regulated by m A modification following export technical noise, or potentially sites that might be bound by into the cytoplasm. These viruses had prominent m A peaks in YTHDF proteins in an m A-independent fashion. A report found NS5, which encodes their viral RNA-dependent RNA polymer- that YTHDF proteins bound to an in vitro transcribed, and ase, strongly suggesting the presence of a conserved RNA reg- hence non-methylated, HCV RNA genome (Rı´os-Marco et al., ulatory element here. Both DENV and ZIKV (PR2015) contained 2016). Therefore, future studies could reveal functions of the m A peaks within their envelope genes, similar to HCV, and 6 6 YTHDF proteins that are independent of m A during the HCV future studies to determine whether these m A sites also affect life cycle. production of infectious flaviviral particles will be of interest. To discern the function of an m A site on HCV RNA during While the genomic RNA structures for DENV, YFV, ZIKV, and infection, we abrogated m A modification in the E1 region of WNV have not yet been determined, these viral genomes contain mut HCV by mutation. This E1 virus produced higher viral titers specific RNA regulatory structures, especially within their UTRs. than the WT virus (Figure 5), similar to what we found with We found that two of the mosquito-transmitted viruses, DENV 6 0 METTL3+14 and YTHDF depletion and suggesting a conserved and YFV, have m A within their 3 UTRs (Figure 6F). In DENV, regulatory mechanism between both m A and the YTHDF pro- the 3 UTR has two stem loops that regulate mosquito to human teins at this site. The presence of these mutations in E1 transmission (Villordo et al., 2015). Therefore, it is possible that increased HCV RNA binding to Core protein while reducing m A patterns and functionality in the mosquito-transmitted flavi- binding to YTHDF2. This suggests that interactions of the HCV viral genomes could contribute to vector-borne transmission. 6 6 RNA with Core are regulated by m A such that viral genomes Finally, we observed clear differences in m A patterns between lacking m A in the E1 region are preferentially segregated for the Dakar and the Puerto Rican isolates of ZIKV, which represent packaging into nascent virions. Therefore, we hypothesize that the African and the Asian lineages, respectively (Haddow et al., the presence or absence of m A in E1 facilitates competition be- 2012). Because these lineages have differences in human dis- tween YTHDF protein and HCV Core binding to the viral genome, ease, with increased pathogenicity ascribed to the Asian lineage leading to the cellular retention or packaging of HCV RNA, of ZIKV (Weaver et al., 2016), the differences in regulation of respectively. these viruses by m A could contribute to these varied infection Because RNA viruses can rapidly evolve under selection pres- outcomes. 6 6 sure, the maintenance of m A sites on the HCV genome sug- In summary, we present global m A profiling of RNA viruses gests that m A must confer an evolutionary advantage to the within the Flaviviridae family. In addition, we provide evidence virus. In HCV, whose pathology is characterized by chronic pro- that an exclusively cytoplasmic RNA is modified by m A. Further- gression during infection in the liver, a slower replication rate has more, we present a role of this modification in regulating HCV been linked to persistent infection through an evasion of immune RNA function at the level of infectious viral particle production. 6 6 surveillance (Bocharov et al., 2004). Therefore, m A may boost This work sets the stage to broadly study the role of m Ain Fla- viral fitness by allowing HCV to establish slow, persistent infec- viviridae infection, transmission, and pathogenesis. This work tions. Pirakitikulr et al. (2016) identified a conserved stem loop also has the potential to uncover regulatory strategies to inhibit in the E1 coding region, just downstream of our identified m A replication by these established and emerging viral pathogens. sites, that suppresses viral particle production without affecting viral RNA replication. This raises the possibility that within the E1 EXPERIMENTAL PROCEDURES region, multiple RNA elements, including m A, play a role in segregating the RNA genome between stages of the HCV life Cell Lines cycle. Human hepatoma Huh7, Huh7.5, and Huh7.5 CD81 KO cells were grown in The function of the other m A sites on the HCV RNA genome DMEM (Mediatech) supplemented with 10% fetal bovine serum (HyClone), remains unknown. Because many of these sites do not overlap 2 5 mM HEPES, and 13 non-essential amino acids (complete [c]DMEM; with YTHDF protein-binding sites, they may directly modify Thermo Fisher Scientific). HCV-K2040 (1B) replicon cells (Wang et al., 2003) were cultured in cDMEM containing 0.2 mg/mL geneticin (Thermo Fisher Sci- HCV RNA structure or recruit alternative m A readers, such as entific). The identity of the Huh7 and Huh7.5 cell lines was verified using the HNRNPA1/B2, eIF3, or even METTL3 (Alarco´ n et al., 2015; Lin Promega GenePrint STR kit (DNA Analysis Facility, Duke University), and cells et al., 2016; Meyer et al., 2015). They may also contribute to anti- were verified as mycoplasma free by the LookOut Mycoplasma PCR detection viral innate immune evasion, because the presence of m Aon kit (Sigma). Huh7.5 CD81 KO cells were generated by CRISPR, as described RNA has been shown to reduce its activation of toll-like receptor before, with details given in the Supplemental Experimental Procedures (Hop- 3 signaling (Kariko´ et al., 2005). While we did not identify m Ain craft et al., 2016; Hopcraft and Evans, 2015). 662 Cell Host & Microbe 20, 654–665, November 9, 2016 Viral Infections and Generation of Viral Stocks Additional experimental procedures can be found in the Supplemental HCV Experimental Procedures. Infectious stocks of a cell culture-adapted strain of genotype 2A JFH1 HCV were generated and titrated by focus-forming assay (FFA), as described ACCESSION NUMBERS (Aligeti et al., 2015). HCV infections were performed at an MOI of 0.3 for 72 hr unless noted. The accession number for the raw sequencing data obtained from the MeRIP- WNV seq and PAR-CLIP and reported in this paper is GEO: GSE83438. Working stocks of WNV isolate TX 2002-HC (WNV-TX) were generated in BHK- 21 cells and titered as described (Suthar et al., 2010). WNV infections (MOI 5) SUPPLEMENTAL INFORMATION were performed in Huh7 cells for 48 hr. DENV and YFV Supplemental Information includes Supplemental Experimental Procedures, Preparation and titering of DENV2-NGC and YFV-17D stocks has been six figures, and three tables and can be found with this article online at described (Le Sommer et al., 2012; Sessions et al., 2009). DENV and YFV in- http://dx.doi.org/10.1016/j.chom.2016.09.015. fections (MOI 2) were performed for 24 hr in Huh7 cells. ZIKV AUTHOR CONTRIBUTIONS ZIKV_PR2015 (PRVABC59) stocks were prepared and titered as described (Quicke et al., 2016). ZIKV_DAK (Zika virus/A.africanus-tc/SEN/1984/41525- N.S.G., A.B.R.M., M.J.M., A.E.R., E.M.K., C.E.M., and S.M.H. designed exper- DAK) stocks were generated and titered by FFA in Vero cells (Le Sommer iments and analyzed the data. N.S.G., A.B.R.M., M.J.M., E.M.K., A.E.R., C.V., et al., 2012). ZIKV infections (MOI 2) were performed in Huh7 cells for 24 hr. J.W., J.A.G., S.E.H., K.M.Q., B.A.L., O.R.I., S.B.B., and S.M.H. performed the experiments. C.L.H., M.J.E., M.S.S., and M.A.G.-B. provided reagents. FFA for HCV Titer N.S.G., A.B.R.M., C.E.M., and S.M.H. wrote the manuscript. All authors Supernatants were collected and filtered through 0.45 mM syringe filters. Serial contributed to editing. dilutions of supernatants were used to infect naive Huh7.5 cells in triplicate wells of a 48-well plate. At either 48 or 72 hpi, cells were fixed, permeabilized, ACKNOWLEDGMENTS and immunostained with HCV NS5A antibody (1:500; gift of Charles Rice, Rockefeller University). Following binding of horseradish peroxidase (HRP)- We thank Dr. Lemon and Dr. Weeks (University of North Carolina-Chapel Hill) conjugated secondary antibody (1:500; Jackson ImmunoResearch), infected and Dr. Rice (Rockefeller University) for reagents; the Duke University Light Mi- foci were visualized with the VIP Peroxidase Substrate Kit (Vector Labora- croscopy Core Facility; the Epigenomics Core Facility at Weill Cornell; and tories) and counted at 403 magnification. Titer (in focus-forming units per members of the S.M.H. and C.E.M. labs for discussion and reading of this milliliter) was calculated as described (Gastaminza et al., 2006). To measure manuscript. This work was supported by funds from the NIH: R01AI125416 intracellular HCV titer, cells pellets were washed in PBS, resuspended in (S.M.H. and C.E.M.); 5P30AI064518 (S.M.H.); T32-CA009111 (A.E.R.); serum-free media, and subjected to five rounds of freezing and thawing in a R25EB020393, R01NS076465, and R01ES021006 (C.E.M.); R01AI089526 dry ice and ethanol bath. Lysate was cleared by centrifugation, and FFA was and R01AI101431 (M.A.G.-B.); R01DK0951250 (M.J.E.); and U19AI083019 performed as described earlier. and R56AI110516 (M.S.S.). Additional funding sources were the Duke White- head Scholarship (S.M.H.), the Ford Foundation (C.V.), the Tri-Institutional MeRIP-Seq Training Program in Computational Biology and Medicine (A.B.R.M.), STARR Poly(A)+ RNA purified from at least 75 mg total RNA (Poly(A) Purist Mag kit; (I7-A765 and I9-A9-071; C.E.M.), the Irma T. Hirschl and Monique Weill- Thermo Fisher Scientific) extracted from HCV-, DENV-, YFV-, WNV-, ZIKV Caulier Charitable Trusts, the Bert L. and N. Kuggie Vallee Foundation, World- (DAK)-, and ZIKV (PR2015)-infected samples was fragmented using the Am- Quant, the Pershing Square Sohn Cancer Research Alliance, NASA bion RNA fragmentation reagent and purified by ethanol precipitation. Frag- (NNX14AH50G and 15-15Omni2-0063), the Bill and Melinda Gates Foundation mented RNA was heated to 75 C for 5 min, placed on ice for 3 min, and (OPP1151054), and the Alfred P. Sloan Foundation (G-2015-13964), the U-TX then incubated with anti-m A antibody (5 mg; Synaptic Systems, #202111) STARs Award (M.A.G.-B.), UTMB (M.A.G.-B. and S.S.B.), Pew Charitable conjugated to Protein G Dynabeads (50 mL; Thermo Fisher Scientific) in MeRIP Trusts (USPHS-AI07647 and ACS-RSG-12-176-01-MPC; M.J.E.), and the buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, and 0.1% NP-40) Burroughs Wellcome Fund. overnight at 4 C. Beads were then washed 53 with MeRIP buffer, and bound RNA was eluted in MeRIP buffer containing 6.7 mM m A sodium salt (Sigma). Received: June 17, 2016 Eluted RNA was purified with the Quick-RNA miniprep kit (Zymo Research) Revised: August 31, 2016 and concentrated by ethanol precipitation. Sequencing libraries were pre- Accepted: September 28, 2016 pared from this RNA, as well as input RNA, using the TruSeq RNA sequencing Published: October 20, 2016 (RNA-seq) kit (Illumina). 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