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Horner, S. M.

Publications and source records attributed to Horner, S. M..

At least 19 recordsLinked to original sources
bioRxiv2026

Modulator Resolves Transcript-, Allele-, and Condition-associated RNA Modification Differences from Direct RNA Sequencing

Nanopore direct-RNA sequencing (DRS) reports RNA modifications on single molecules, but there is no comprehensive algorithm for processing, aligning, and differentiating base modifications across reads that are often 5'-truncated. Here we present Modulator, which assigns every read, truncated or not, to a fragmentform, defined by a shared transcript 3'-end and the splice-junctions upstream of it. Of note, Modulator builds fragmentforms from genomic alignments without a reference transcriptome, estimates per-base modification stoichiometry within each fragmentform, and classifies each stoichiometry difference as transcript-, allele-, or condition-dependent. In the control HG002 transcriptome, Modulator built 23,746 fragmentforms, with 15,620 candidate novel isoforms that carried poly(A) signals as often as previously annotated transcripts. Also, assigning 5'-truncated reads to fragmentforms nearly doubled the number of testable and differentially modified sites. m6A sites within 100 nucleotides (nt) of a splice junction or within 1 kb of a poly(A) site were less modified in fragmentforms that use that junction or poly(A) site, indicating that local transcript context shapes where m6A is deposited. From the same reads, Modulator called SNPs with 93% precision and no phasing errors against the Genome in a Bottle benchmark. It identified 131 SNPs that create or remove a modifiable nucleotide and 1,358 allele-specific modification differences, most involving SNPs more than 50 nt from the modified site. In cells infected with Zika virus, which remodels gene expression relative to uninfected cells, Modulator identified 1,253 transcript-independent modification stoichiometry changes. Of the m6A changes at sites measured by GLORI, 96% agreed in direction with single-nucleotide-resolution GLORI maps of the same samples. Thus, Modulator accurately separates changes in RNA modification from transcript-associated and allele-associated differences and can improve the utility of nanopore DRS. Availability and Implementation: Modulator is free and open source: https://github.com/Theo-Nelson/Modulator. It is written in Python and runs on Linux OS.

bioinformatics↗
bioRxiv2026

The m6A methyltransferase complex protein VIRMA engages hepatitis C virus RNA through a WTAP-independent mechanism

Cellular proteins in the m6A methyltransferase protein complex (m6A-MTC) work together to modify both cellular and viral RNAs with N6-methyladenosine (m6A), an RNA modification that regulates viral infection. We previously showed that hepatitis C virus (HCV), a cytoplasmic positive-sense RNA virus, has an m6A-modified RNA genome. We also found that m6A deposition on HCV RNA requires WTAP, which promotes association of the METTL3-METTL14 catalytic heterodimer to the viral RNA. However, the factors that directly recognize HCV RNA and recruit the m6A-MTC to the viral genome are unknown. Here, we identify the cellular m6A-MTC component VIRMA as an HCV RNA-targeting factor required for METTL3 association with the viral genome. We show that endogenous VIRMA can associate with both HCV and cellular RNAs independently of WTAP. Further, we define distinct domains of VIRMA involved in WTAP interaction and RNA binding and show that the regions of VIRMA involved in WTAP-interaction are required for cellular but not HCV RNA association. Together, these results establish that VIRMA uses distinct molecular determinants to engage viral and cellular RNAs and define VIRMA as a substrate-recognition factor required for m6A-MTC association with HCV RNA.

molecular biology↗
bioRxiv2026

Alternative polyadenylation drives isoform-dependent m6A remodeling during Zika virus infection

Alternative RNA processing generates extensive transcript diversity, yet how transcript architecture influences selective m6A deposition is incompletely understood. Exon-junction-based models explain where m6A is excluded, but a positive determinant of m6A accumulation remains undefined. Here, we leverage Zika virus-induced changes in m6A deposition to uncover determinants of transcript-selective methylation. By integrating GLORI-seq, native METTL3 RNA immunoprecipitation, and nanopore direct RNA sequencing, we generate a single-nucleotide, isoform-resolved map of m6A dynamics during infection. We identify over 2,000 dynamic m6A sites, many arising from changes in transcript architecture, and pinpoint proximal polyadenylation sites as positive determinants of m6A accumulation. The cleavage stimulation factors CSTF2 and CSTF2T drive this remodeling through two routes: redundant induction of intronic polyadenylation, which converts internal exons into terminal exons that expose DRACH motifs to METTL3, and non-redundant, cleavage-independent recruitment of METTL3 near proximal polyadenylation sites, establishing alternative polyadenylation as a key architectural determinant of the m6A landscape.

molecular biology↗
bioRxiv2026

IFIT3 Associates with m⁶A-Modified RNA to Restrict Hepatitis C Virus Infection

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are RNA-binding effectors that restrict infection by diverse RNA viruses. Among the IFIT family, how IFIT3 recognizes RNA remains the least understood. Here, we identify IFIT3 as preferentially associating with N6-methyladenosine (mA)-modified hepatitis C virus (HCV) genomic RNA and host transcripts to restrict HCV infection. IFIT3 cellular RNA binding sites and mA sites, mapped transcriptome-wide by HyperTRIBE-seq during HCV infection, showed significant overlap. This mA preference was further supported by findings that IFIT3 binding sites significantly overlapped those of established mA-binding proteins; that inhibiting mA installation reduced IFIT3 association with mA-modified HCV RNA and cellular transcripts; that IFIT3 co-purified more efficiently with mA-modified short RNA probes than with unmodified controls; and that mutating mA consensus motifs in the HCV genome reduced IFIT3 association with viral RNA. Structure-function analyses identified two regions required for RNA probe binding: tetratricopeptide repeat domains 1-2 (TPR1-2) and a previously uncharacterized predicted helical hairpin between TPRs 6 and 7. In infected cells, the helical hairpin was required for IFIT3 association with HCV RNA but dispensable for interactions with other IFIT proteins. Conversely, TPR1-2 was dispensable for HCV RNA binding but essential for IFIT2 interaction, establishing that these functions are structurally separable. Loss of either region diminished antiviral activity, as indicated by increased levels of HCV RNA in clarified supernatants. Consistent with models of mA-linked restriction of late stages of infection, extracellular HCV RNA showed reduced mA and decreased IFIT3 association relative to intracellular RNA. Together, these findings define an mA-linked mechanism by which IFIT3 engages viral RNA and reveal an unexpected role for mA in antiviral effector function. SIGNIFICANCERNA-binding proteins are critical effectors of antiviral defense, yet for many of these proteins the mechanisms of viral RNA recognition remain unclear. Here, we show that m6A modification on both host and viral RNA promotes recognition by the interferon stimulated gene, IFIT3. RNA recognition did not require interaction with IFIT1 or IFIT2, although IFIT3 antiviral function required both RNA binding and interaction with IFIT2. These findings identify m6A as a new regulator of IFIT protein function and broaden our understanding of how RNA modifications shape antiviral restriction.

immunology↗
bioRxiv2025

A small cationic probe for accurate, punctate discovery of RNA tertiary structure

RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively-charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to non-bridging phosphate oxygens, creating localized regions of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.

biochemistry↗
bioRxiv2025

14-3-3ϵ UFMylation promotes RIG-I-mediated signaling

Post-translational modifications regulate RIG-I signaling in diverse ways. We previously showed that UFMylation, the covalent attachment of the ubiquitin-fold modifier UFM1 to proteins, enhances RIG-I signaling by promoting its interaction with its membrane-targeting adaptor 14-3-3{varepsilon}. Here, we map UFM1 conjugation to lysines K50 and K215 on 14-3-3{varepsilon} and demonstrate how these UFMylation events control RIG-I signaling. Using in vitro and cellular UFMylation assays, we reveal that K50R/K215R mutations abolish UFMylation and reduce type I and III interferon induction following RIG-I activation. Unexpectedly, these mutations do not disrupt 14-3-3{varepsilon}-RIG-I interaction. Instead, they paradoxically enhance RIG-I interaction with MAVS while simultaneously reducing 14-3-3{varepsilon}-MAVS interaction. These findings establish UFMylation of 14-3-3{varepsilon} as an important control that shapes MAVS complex architecture to ensure optimal RIG-I signaling and highlights the broader regulatory role of UFMylation in antiviral innate immunity. ImportancePost-translational modifications provide regulatory control of antiviral innate immune responses. Our study reveals that UFMylation of 14-3-3{varepsilon} is required for RIG-I-mediated innate immune signaling. We demonstrate that conjugation of UFM1 to specific lysine residues on 14-3-3{varepsilon} enhances downstream signaling events that facilitate interferon induction. It does this by stabilizing 14-3-3{varepsilon} association with the MAVS signaling complex and coordinating productive complex architecture. By identifying the precise sites of UFMylation on 14-3-3{varepsilon} and their functional consequences, we provide insights into the regulatory layers governing antiviral innate immunity. These findings complement emerging evidence that UFMylation serves as a versatile modulator across diverse immune pathways. Furthermore, our work highlights how protein chaperones like 14-3-3{varepsilon} can be dynamically modified to orchestrate complex signaling cascades, suggesting potential therapeutic approaches for targeting dysregulated innate immunity.

immunology↗
bioRxiv2025

UFMylation promotes orthoflavivirus infectious particle production

Post-translational modifications play crucial roles in viral infections, yet many potential modifications remain unexplored in orthoflavivirus biology. Here we demonstrate that the UFMylation system, a post-translational modification system that catalyzes the transfer of UFM1 onto proteins, promotes infection by multiple orthoflaviviruses including dengue virus, Zika virus, West Nile virus, and yellow fever virus. We found that depletion of the UFMylation E3 ligase complex proteins UFL1 and UFBP1, as well as other UFMylation machinery components (UBA5, UFC1, and UFM1), significantly reduces infectious virion production for orthoflaviviruses but not the hepacivirus, hepatitis C. Mechanistically, UFMylation does not regulate viral RNA translation or RNA replication but instead affects a later stage of the viral lifecycle. We identified novel interactions between UFL1, and several viral proteins involved in orthoflavivirus virion assembly, including NS2A, NS2B-NS3, and Capsid. These findings establish UFMylation as a previously unrecognized post-translational modification system that promotes orthoflavivirus infection, likely through modulation of viral assembly. This work expands our understanding of the post-translational modifications that control orthoflavivirus infection and identifies new potential therapeutic targets. ImportanceOrthoflaviviruses depend on host-mediated post-translational modifications to successfully complete their lifecycle, yet many of these critical interactions remain undefined. Here, we describe a role for a post-translational modification pathway, UFMylation, in promoting infectious particle production of ZIKV and DENV. We show that UFMylation regulates these viruses at a lifecycle stage after initial RNA translation and RNA replication. Additionally, we find that regulation of infection by UFMylation extends to other orthoflaviviruses, including West Nile virus and yellow fever virus, but not to the broader Flaviviridae family. Finally, we demonstrate that UFMylation machinery directly interacts with specific DENV and ZIKV proteins during infection. These studies reveal a previously unrecognized role for UFMylation in regulating orthoflavivirus infection.

microbiology↗
bioRxiv2023

Cellular RNA interacts with MAVS to promote antiviral signaling

Immune signaling needs to be well-regulated to promote clearance of pathogens, while preventing aberrant inflammation. Interferons (IFNs) and antiviral genes are activated by the detection of viral RNA by RIG-I-like receptors (RLRs). Signal transduction downstream of RLRs proceeds through a multi-protein complex organized around the central adaptor protein MAVS. Recent work has shown that protein complex function can be modulated by RNA molecules providing allosteric regulation or acting as molecular guides or scaffolds. Thus, we hypothesized that RNA plays a role in organizing MAVS signaling platforms. Here, we show that MAVS, through its central intrinsically disordered domain, directly interacts with the 3' untranslated regions of cellular mRNAs. Importantly, elimination of RNA by RNase treatment disrupts the MAVS signalosome, including newly identified regulators of RLR signaling, and inhibits phosphorylation of the transcription factor IRF3. This supports the hypothesis that RNA molecules scaffold proteins in the MAVS signalosome to induce IFNs. Together, this work uncovers a function for cellular RNA in promoting signaling through MAVS and highlights a generalizable principle of RNA regulatory control of cytoplasmic immune signaling complexes.

immunology↗
bioRxiv2022

Structure-first identification of conserved RNA elements that regulate dengue virus genome architecture and replication.

The genomes of RNA viruses encode the information required for replication in host cells in both their linear sequence and in complex higher-order structures. A subset of these complex functional RNA genome structures show clear sequence conservation. However, the extent to which viral RNA genomes contain conserved structural elements - that cannot be detected by sequence alone - that nonetheless are critical to viral fitness is largely unknown. Here, we take a structure-first approach to identify motifs conserved across the coding sequences of the RNA genomes for the four dengue virus (DENV) serotypes. We used SHAPE-MaP to identify 22 candidate motifs with conserved RNA structures, but no prior association with viral replication. At least ten of these motifs are important for viral fitness, revealing a significant unnoticed extent of RNA structure-mediated regulation within viral coding sequences. These conserved viral RNA structures promote a compact global genome architecture, interact with proteins, and regulate the viral replication cycle. These motifs are constrained at the levels of both RNA structure and protein sequence and are potential resistance-refractory targets for antivirals and live-attenuated vaccines. Structure-first identification of conserved RNA structure is poised to guide efficient discovery of RNA-mediated regulation in viral genomes and other cellular RNAs.

biochemistry↗
bioRxiv2022

WTAP targets the METTL3 m6A-methyltransferase complex to cytoplasmic hepatitis C virus RNA to regulate infection

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral lifecycle. This lifecycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and for m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production were independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCEPositive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described how HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV lifecycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic lifecycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

microbiology↗
bioRxiv2021

Signaling from the RNA sensor RIG-I is regulated by ufmylation

The RNA binding protein RIG-I is a key initiator of the antiviral innate immune response. The signaling that mediates the antiviral response downstream of RIG-I is transduced through the adaptor protein MAVS and results in the induction of type I and III interferons (IFN). This signal transduction occurs at endoplasmic reticulum (ER)-mitochondrial contact sites, to which RIG-I and other signaling proteins are recruited following their activation. RIG-I signaling is highly regulated to prevent aberrant activation of this pathway and dysregulated induction of IFN. Previously, we identified UFL1, the E3 ligase of the ubiquitin-like modifier conjugation system called ufmylation, UFL1, as one of the proteins recruited to membranes at ER-mitochondrial contact sites in response to RIG-I activation. Here, we show that UFL1, as well as the process of ufmylation, promote IFN induction in response to RIG-I activation. We find that following RNA virus infection, UFL1 is recruited to the membrane targeting protein 14-3-3{varepsilon}, and that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling. Importantly, we found that 14-3-3{varepsilon} has an increase in UFM1-conjugation following RIG-I activation. Additionally, loss of cellular ufmylation prevents the interaction of 14-3-3{varepsilon} with RIG-I, which abrogates the interaction of RIG-I with MAVS and thus downstream signal transduction that induces IFN. Our results define ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a post-translational control for IFN induction. SignificanceThe viral RNA sensor RIG-I initiates the antiviral innate immune response by activating a signaling cascade that induces interferon. Activation of the RIG-I signaling pathway is highly regulated to quickly mount a protective immune response while preventing dysregulation that can lead to excessive inflammation or autoimmune disorders. Here, we characterize one such mechanism of regulation. We describe that UFL1, an E3 ligase for the ubiquitin-like modifier conjugation system called ufmylation, is important to promote RIG-I signaling. Using molecular approaches, we show that ufmylation promotes RIG-I interaction with the membrane targeting protein 14-3-3{varepsilon}. As such, ufmylation positively regulates RIG-I recruitment to its signaling adaptor proteins MAVS for induction of interferon in response to RNA virus infection.

immunology↗
bioRxiv2020

Post-Transcriptional Regulation of Antiviral Gene Expression by N6-Methyladenosine

Type I interferons (IFN) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. These ISGs require strict regulation for an efficient and controlled antiviral response, but post-transcriptional controls of these genes have not been well defined. Here, we identify a new role for the RNA base modification N6-methyladenosine (m6A) in the regulation of ISGs. Using ribosome profiling and quantitative mass spectrometry, coupled with m6A-immunoprecipitation and sequencing, we identified a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determined that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A binding-dependent manner. Importantly, we found that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as a post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction.

immunology↗
bioRxiv2020

The mRNA cap 2'O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes

Type I interferons (IFN) initiate an antiviral state through a signal transduction cascade that leads to the induction of hundreds of IFN-stimulated genes (ISGs) to restrict viral infection. Recently, RNA modifications on both host and viral RNAs have been described as regulators of infection. However, the impact of host mRNA cap modifications on the IFN response and how this regulates viral infection is unknown. Here, we reveal that CMTR1, an ISG that catalyzes 2O methylation of the first transcribed nucleotide in cellular mRNA (Cap 1), promotes the protein expression of specific ISGs that contribute to the antiviral response. Depletion of CMTR1 reduces the IFN-induced protein levels of ISG15, MX1, and IFITM1, without affecting their transcript abundance. However, CMTR1 depletion does not significantly affect the IFN-induced protein or transcript abundance of IFIT1 and IFIT3. Importantly, knockdown of IFIT1, which acts with IFIT3 to inhibit the translation of RNAs lacking Cap 1 2O methylation, restores protein expression of ISG15, MX1, and IFITM1 in cells depleted of CMTR1. Finally, we found that CMTR1 plays a role in restricting RNA virus replication, likely by ensuring the expression of specific antiviral ISGs. Taken together, these data reveal that CMTR1 is required to establish an antiviral state by ensuring the protein expression of a subset of ISGs during the type I IFN response. ImportanceInduction of an efficient type I IFN response is important to control viral infection. We show that the host 2O methyltransferase CMTR1 facilitates the protein expression of ISGs in human cells by preventing IFIT1 from inhibiting the translation of these mRNAs lacking cap 2O methylation. Thus, CMTR1 promotes the IFN-mediated antiviral response.

immunology↗
bioRxiv2019

Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, we profiled m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.

molecular biology↗
bioRxiv2019

Altered m6A modification of specific cellular transcripts affects Flaviviridae infection

The RNA modification N6-methyladenosine (m6A) can modulate mRNA fate and thus affect many biological processes. We analyzed m6A modification across the transcriptome following infection by dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and hepatitis C virus (HCV). We found that infection by these viruses in the Flaviviridae family alters m6A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m6A to RIOK3 promotes its translation, while loss of m6A in CIRBP promotes alternative splicing. Importantly, we found that activation of innate immune sensing or the endoplasmic reticulum (ER) stress response by viral infection contributes to the changes in m6A modification in RIOK3 and CIRBP, respectively. Further, several transcripts with infection-altered m6A profiles, including RIOK3 and CIRBP, encode proteins that influence DENV, ZIKV, and HCV infection. Overall, this work reveals that cellular signaling pathways activated during viral infection lead to alterations in m6A modification of host mRNAs to regulate infection.

molecular biology↗
bioRxiv2019

Limits in the detection of m6A changes using MeRIP/m6A-seq

Many cellular mRNAs contain the modified base m6A, and recent studies have suggested that various stimuli can lead to changes in m6A. The most common method to map m6A and to predict changes in m6A between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m6A peak overlap in mRNAs varies from [~]30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m6A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes.

bioinformatics↗
bioRxiv2019

Hepatitis C virus infection is inhibited by a non-canonical antiviral signaling pathway targeted by NS3-NS4A

The hepatitis C virus (HCV) NS3-NS4A protease complex is required for viral replication and is the major viral innate immune evasion factor. NS3-NS4A evades antiviral innate immunity by inactivating several proteins, including MAVS, the signaling adaptor for RIG-I and MDA5, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Here, we identified a Tyr-16-Phe (Y16F) change in the NS4A transmembrane domain that prevents NS3-NS4A targeting of Riplet but not MAVS. This Y16F substitution reduces HCV replication in Huh7 cells, but not in Huh-7.5 cells, known to lack RIG-I signaling. Surprisingly, deletion of RIG-I in Huh7 cells did not restore Y16F viral replication. Rather, we found that Huh-7.5 cells lack Riplet expression and that addition of Riplet to these cells reduced HCV Y16F replication. In addition, IRF3 deletion in Huh7 cells was sufficient to restore HCV Y16F replication, and the Y16F protease lacked the ability to prevent IRF3 activation or interferon induction. Taken together, these data reveal that the NS4A Y16 residue regulates a non-canonical Riplet-IRF3-dependent, but RIG-I-MAVS-independent, signaling pathway that limits HCV infection.\n\nImportanceThe HCV NS3-NS4A protease complex facilitates viral replication by cleaving and inactivating the antiviral innate immune signaling proteins MAVS and Riplet, which are essential for RIG-I activation. NS3-NS4A therefore prevents IRF3 activation and interferon induction during HCV infection. Here, we uncover an amino acid residue within the NS4A transmembrane domain that is essential for inactivation of Riplet, but does not affect MAVS cleavage by NS3-NS4A. Our study reveals that Riplet is involved in a RIG-I- and MAVS-independent signaling pathway that activates IRF3 and that this pathway is normally inactivated by NS3-NS4A during HCV infection. Our study selectively uncouples these distinct regulatory mechanisms within NS3-NS4A and defines a new role for Riplet in the antiviral response to HCV. As Riplet is known to be inhibited by other RNA viruses, such as such influenza A virus, this innate immune signaling pathway may also be important in controlling other RNA virus infections.

microbiology↗