Browsing by Author "Horner, Stacy M"
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Item Open Access An Atlas of Genetic Variation Linking Pathogen-Induced Cellular Traits to Human Disease.(Cell host & microbe, 2018-08) Wang, Liuyang; Pittman, Kelly J; Barker, Jeffrey R; Salinas, Raul E; Stanaway, Ian B; Williams, Graham D; Carroll, Robert J; Balmat, Tom; Ingham, Andy; Gopalakrishnan, Anusha M; Gibbs, Kyle D; Antonia, Alejandro L; eMERGE Network; Heitman, Joseph; Lee, Soo Chan; Jarvik, Gail P; Denny, Joshua C; Horner, Stacy M; DeLong, Mark R; Valdivia, Raphael H; Crosslin, David R; Ko, Dennis CPathogens have been a strong driving force for natural selection. Therefore, understanding how human genetic differences impact infection-related cellular traits can mechanistically link genetic variation to disease susceptibility. Here we report the Hi-HOST Phenome Project (H2P2): a catalog of cellular genome-wide association studies (GWAS) comprising 79 infection-related phenotypes in response to 8 pathogens in 528 lymphoblastoid cell lines. Seventeen loci surpass genome-wide significance for infection-associated phenotypes ranging from pathogen replication to cytokine production. We combined H2P2 with clinical association data from patients to identify a SNP near CXCL10 as a risk factor for inflammatory bowel disease. A SNP in the transcriptional repressor ZBTB20 demonstrated pleiotropy, likely through suppression of multiple target genes, and was associated with viral hepatitis. These data are available on a web portal to facilitate interpreting human genome variation through the lens of cell biology and should serve as a rich resource for the research community.Item Open Access Characterizing novel molecular regulators of antiviral gene expression(2020) McFadden, Michael JThe intracellular innate immune response to viral infection is among the first lines of defense against these pathogens. For the early establishment of an antiviral cellular state and initiation of inflammatory responses, type I interferons (IFNs) are particularly important, as they potently induce the production of hundreds of IFN-stimulated genes (ISGs), many of which have antiviral functions. The type I IFN response requires tight molecular coordination to achieve both efficient production of antiviral proteins and controlled shutoff of inflammatory responses to avoid tissue damage and autoimmunity. Despite the importance of regulation of this antiviral response, current knowledge of the molecular controls governing its activation and suppression remains incomplete. Further, although ISGs have diverse functions and are induced to differing potencies, our understanding of regulatory controls governing the expression of individual or subclasses of ISGs is limited. Current knowledge of type I IFN response regulation is predominantly centered on transcriptional and post-translational regulatory controls. However, post-transcriptional regulation of antiviral responses has begun to emerge as an important layer of control. An example of these post-transcriptional regulatory controls is the RNA base modification N6-methyladenosine (m6A), which regulates many aspects of mRNA metabolism through transcript-specific effects. m6A deposition is mediated by a cellular complex of proteins including METTL3 and METTL14 (METTL3/14) and other cofactors, and m6A can also be removed from RNA by the demethylase proteins FTO and ALKBH5. The presence of m6A on viral and host RNAs has been shown to influence the outcome of infection by diverse viruses. However, the role of m6A in the response to type I IFNs has not been explored. To investigate the role of m6A in the type I IFN response, we began by manipulating m6A levels in the transcriptome through perturbation of the expression of the cellular m6A machinery and measuring the induction of ISGs after IFN treatment. We found that depletion of the m6A methyltransferase proteins METTL3 and METTL14 (METTL3/14) resulted in less protein production of a subset of ISGs, including the antiviral genes IFITM1 and MX1, after IFN treatment. However, the expression of other ISGs and the overall activation of the IFN responses were unchanged. Using methyl RNA immunoprecipitation and sequencing (meRIP-seq), we found that the transcripts of many ISGs are modified by m6A, and these included the METTL3/14-regulated ISGs IFITM1 and MX1 that we had identified. Using polysome profiling and ribosome profiling, we determined that METTL3/14-regulated ISGs are translationally enhanced by METTL3/14. Additionally, ablation of putative m6A sites within the 3’UTR of IFITM1 decreased the translation of a reporter molecule. Overexpression of the m6A reader protein YTHDF1, which has known roles in promoting translation, enhanced the expression of IFITM1 in an m6A binding-dependent fashion. These experiments characterized METTL3/14 and m6A as novel enhancers of the type I IFN response. To determine whether m6A contributes to type I IFN-mediated viral restriction, we depleted or overexpressed METTL3/14 and pretreated cells with a low dose of IFN-β prior to infection with vesicular stomatitis virus (VSV). Interestingly, METTL3/14 depletion decreased the expression of ISGs and allowed increased VSV infection, while METTL3/14 overexpression had the opposite effect. Together, these studies demonstrate that METTL3/14 and m6A enhance the antiviral effect of type I IFN by promoting the translation of ISGs to support the establishment of an antiviral cellular state. Having discovered a role for m6A in the type I IFN response, we also investigated the role of an m6A demethylase protein, FTO. FTO polymorphisms can have profound effects on human health. Certain polymorphisms are associated with fat mass and obesity, cardiovascular disease, while others can cause growth retardation or embryonic lethality. However, the molecular functions of FTO and the cellular pathways that it affects are still not well characterized. We depleted FTO and measured the production of ISGs following IFN-β treatment and found that the production of m6A-regulated ISGs was increased, as expected. However, unexpectedly, we found that FTO depletion increased the mRNA levels of a subset of ISGs. Pulse labeling of nascent transcripts revealed that FTO suppresses the transcription of these ISGs and that FTO-depleted cells are primed for the production of certain ISGs in response to IFN. We then used cells lacking PCIF1, the writer of 2’-O-N6-dimethyladenosine (m6Am), an RNA modification that FTO can also remove, and found that FTO-mediated regulation of ISGs occurs independently of the m6Am modification. These results identify FTO as a transcriptional regulator of a subset of ISGs, which will add an important dimension to our understanding of the molecular functions of FTO and its contributions to inflammatory disease. Future research revealing the mechanisms by which FTO suppresses ISG transcription will be of great interest. Together, these data identify novel functions of m6A and its related cellular machinery in both positive and negative regulation of the type I IFN response and antiviral gene expression.
Item Open Access Cytoplasmic N6-Methyladenosine Deposition on Hepatitis C Viral RNA(2022) Sacco, Matthew TylerHepatitis C virus (HCV) exploits nearly all aspects of cellular RNA biology to regulate its viral RNA genome during infection. However, the molecular mechanisms by which HCV exploits one aspect of RNA regulation, RNA modification with N6-methyladenosine (m6A), are still emerging. This is because the current understanding of how RNA becomes m6A modified involves a variety of nuclear localized mechanisms that are incompatible with the cytoplasmic lifecycle of HCV. Thus, we set out to bridge the gap between our current understanding of m6A biology and how HCV RNA becomes m6A modified.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.
Item Open Access N6-methyladenosine (m6A) at the RNA virus-host interface(2019) Gokhale, Nandan SatishRNA is a versatile and tractable biomolecule that serves as a critical component of life, whether as a script for protein production, a carrier of genetic information, a scaffold, or an enzyme. The fate and function of RNA can be influenced by chemical modifications such as N6-methyladenosine (m6A). Here we sought to identify the role of m6A during infection by positive-sense RNA viruses in the Flaviviridae family.
First, we investigated the role of m6A on viral RNA. We mapped m6A on the viral RNA genomes of hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and yellow fever virus (YFV). We then studied HCV as a model RNA and virus to understand the function of m6A on Flaviviridae RNA genomes. We found that the m6A methyltransferases METTL3 and METTL14 reduced HCV infectious particle production without affection viral RNA replication, while the m6A demethylase FTO had the opposite effect. Similarly, the m6A-binding YTHDF1-3 proteins also inhibited HCV particle production. Furthermore, the YTHDF proteins relocalized to cytoplasmic lipid droplets, the sites of HCV particle assembly, during infection. We then identified that m6A in a specific region of viral RNA was responsible for the role of m6A in viral particle production. Abrogation of m6A modification increased viral RNA binding to the capsid protein Core, an important step of HCV assembly, and also increased HCV particle production. These data suggest that m6A inhibits HCV particle production and that m6A modification of viral RNA can have a functional consequence for infection.
We then investigated how m6A on cellular mRNA can impact Flaviviridae infection. Working in collaboration with Dr. Chris Mason’s lab, we developed stringent analytical tools for detecting m6A changes. When we applied these tools, we found that a subset of cellular transcripts had altered m6A modification following infection by DENV, ZIKV, WNV, and HCV. We identified that innate immune signaling and ER stress, cellular pathways which are activated during Flaviviridae infection, contribute to altered m6A modification of two model transcripts RIOK3 and CIRBP. The gain of m6A on RIOK3 promotes the translation of this transcript, while loss of m6A on CIRBP influences its alternative splicing. Importantly, the RIOK3, CIRBP, and other transcripts with altered m6A modification can promote or inhibit Flaviviridae infection. Taken together, these results highlight the important role of m6A on both viral and cellular RNA in regulating infection.
Item Open Access New Insights into Hepatitis C Virus Assembly and Envelopment(2018) Roder, AllisonHepatitis C virus assembly and envelopment are coordinated by a complex network of protein-protein interactions that encompasses most of the viral structural and nonstructural proteins. However, the details surrounding how these proteins function during the late stages of the viral lifecycle remain unclear. Here, we define new mechanisms for three viral proteins, p7, NS5B and NS4A, in viral assembly and envelopment.
First, we characterized a culture adapted genotype 2A, JFH-1, strain of HCV that showed a 2-log increase in viral titer after more than 30 passages in cell culture. Sequencing of the adapted virus revealed 8 synonymous and 9 nonsynonymous amino acid changes. We found that two of these mutations, in the p7 and NS5B proteins, together, were sufficient to recapitulate the increase in viral infectivity seen in the fully adapted virus, without increasing intracellular RNA levels. The two mutations were found to promote an increase in cellular lipid droplet size and circularity and ultimately lead to a decrease in sphingomyelin content of infectious virions. These data suggest a genetic interaction between the p7 and NS5B proteins that enhances viral particle production and increases viral specific infectivity.
In the second study presented in this dissertation, site-directed mutagenesis of the C-terminal acidic domain of the viral NS4A protein was used to investigate its function. We found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from WT cells. These results suggest that this Y45F mutation in NS4A causes secretion of low density Core particles devoid of genomic HCV RNA. These results corroborate previous findings showing that mutation of the first hydrophobic region of E1 also causes secretion of Core complexes lacking RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.
Item Open Access Novel mechanisms of antiviral innate immune regulation by the hepatitis C virus NS3-NS4A protease(2019) Vazquez, ChristineHepatitis C virus (HCV) evasion of the host immune system is largely mediated by the actions of the HCV NS3-NS4A protease complex, which consists of the serine protease and RNA helicase NS3 and its membrane targeting co-factor NS4A. NS3-NS4A has multiple functions in the HCV life cycle, with roles in both HCV replication and regulation of innate immune signaling. To regulate innate immune signaling, NS3-NS4A inactivates multiple signaling proteins, including MAVS, an adaptor protein in the RIG-I antiviral signaling pathway, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Inactivation of these host proteins results in an inhibition of downstream signaling through the transcription factor IRF3 and inhibition of the subsequent induction of IFN-β. What directs the multiple functions of NS3-NS4A throughout the HCV life cycle is largely undetermined. Here, we identify a tyrosine residue within the transmembrane domain of NS4A that uncouples the various function of NS3-NS4A.
First, to uncouple the roles of NS3-NS4A in replication and immune evasion, I focused on the NS4A transmembrane domain and generated an NS4A mutant (Y16F) in a full-length HCV infectious clone, a subgenomic replicon, and an over-expression construct. I then assessed viral replication of HCV wild-type (WT) and Y16F viruses by measuring replication of a subgenomic HCV replicon in two related liver hepatoma cell lines: Huh7, which have functional RIG-I signaling, and Huh-7.5 cells, which lack functional RIG-I signaling. The HCV Y16F virus replicated to similar levels as WT HCV in Huh-7.5 cells. However, in Huh7 cells, replication of HCV Y16F was decreased compared to the HCV WT. I used CRISPR-Cas9 gene editing to delete proteins in the RIG-I pathway, including RIG-I, MAVS, and IRF3, in Huh7 cells, infected these cells with HCV WT or Y16F viruses, and then measured virus replication. I found that Y16F viral replication was not restored to the levels of WT in Huh7-RIG-I KO cells or the Huh7-MAVS KO cells, but it was restored to the levels of WT in the Huh7-IRF3 KO cells. I also found that the HCV NS3-NS4A Y16F mutation reduced the ability of over-expressed NS3-NS4A to block IRF3 activation, as measured through nuclear translocation via immunofluorescence microscopy. Further the NS3-NS4A Y16F mutation also had a reduced ability to block the induction of interferon-stimulated genes during both HCV replication and infection. This reveals that HCV NS4A Y16 can regulate a RIG-I-independent, yet IRF3-dependent, signaling pathway that limits viral replication.
Second, to further characterize this RIG-I-independent, IRF3-dependent signaling pathway, I examined the interactions of HCV NS3-NS4A with two of its known host substrates, MAVS and Riplet. To test whether the Y16F mutation prevented NS3-NS4A cleavage of MAVS, I performed a MAVS cleavage assay during both overexpression of NS3-NS4A and MAVS and also during infection with HCV WT and Y16F viruses. NS3-NS4A Y16F was able to cleave MAVS just like WT during both conditions. Next, I found that over-expression of NS3-NS4A WT changed Riplet intracellular localization and that NS4A interacted with Riplet. However, the NS4A Y16F mutation prevented NS4A-Riplet interactions in both of these contexts. Interestingly, I found that Huh-7.5 cells express lower levels of Riplet protein and mRNA compared to Huh7 cells. When full-length Riplet was added exogenously to Huh-7.5 cells, HCV Y16F virus replication was reduced compared to WT. However, when a Riplet construct missing the RING domain, which is essential for Riplet signaling, was added exogenously to Huh-7.5, both WT and Y16F viruses now replicated similarly. Taken together, these data identify NS3-NS4A Y16 as important for regulating a previously uncharacterized Riplet-mediated signaling pathway that limits HCV infection.
Item Open Access Regulation of the Antiviral Innate Immune Response by Ufmylation(2022) Snider, DaltryCellular detection of viral infection activates an antiviral program through the induction of interferons (IFN), which ultimately limit viral replication and reduce viral spread. This induction of IFNs is initiated by proteins that sense viral RNA, such as RIG-I, and proteins that coordinate the resulting signaling, like its adaptor protein MAVS. These proteins, along with others, form a signaling pathway that is highly controlled to provide a fine balance between clearance of viral infection, while preventing prolonged or excessive IFN induction which can lead to autoimmunity. As such, the RIG-I signaling cascade is carefully regulated by multiple mechanisms, including post-translational modifications (PTMs), protein-protein interactions, and protein localization. For example, following sensing of viral RNA, RIG-I is ubiquitinated, leading to its activation and subsequent interaction with the molecular trafficking protein 14-3-3epsilon, forming a RIG-I signaling complex. This interaction with 14-3-3epsilon facilitates the relocalization of activated RIG-I from the cytosol to intracellular membranes where it interacts with the adaptor protein MAVS leading to the propagation of various signals that induce IFN. Importantly, while some post-translational modifications are well studied in regulation of the RIG-I signaling pathway and are known control many aspects of IFN induction, our current knowledge of the full set of regulatory mechanisms that govern the RIG-I activation cascade is incomplete. Previously, to determine novel regulators of RIG-I signaling, we identified proteins that relocalize to the RIG-I/MAVS signaling platform at ER-mitochondrial membrane contact sites. These relocalized proteins include UFL1, the E3 ligase of the small ubiquitin-like modification called UFM1. Similar to ubiquitination, the ufmylation process conjugates a small protein (UFM1) to lysine residues of target proteins using an E1, E2, and E3 ligase machinery system. Further, a protease specific to ufmylation can remove the modification, allowing for dynamic regulation of protein function. However, in contrast to ubiquitin, the ways in which ufmylation regulates protein function is still unclear. While the role of well-described PTMs such as ubiquitination and phosphorylation have a well-respected role in the regulation of the RIG-I signaling pathway, little is known about how emerging ubiquitin-like modifications, such as ufmylation, regulate this antiviral signaling pathway. Indeed, how these newly discovered PTMs regulate protein function has not yet been elucidated, requiring further study. Through investigating how ufmylation may regulate RIG-I signaling, I found that UFL1 is essential for the induction of IFN following RNA virus infection using a combination of IFN reporter assays, RT-qPCR to assess transcription, and immunoblots and ELISA to investigate protein expression. This regulatory role of UFL1 relies on its ability to conjugate UFM1 onto target proteins, leading me to hypothesize that ufmylation of proteins themselves may be driving signaling regulation. Indeed, altered expression of UFM1, or any of the proteins involved in its conjugation also positively regulated IFN. Utilizing co-immunoprecipitation assays, I found that following RNA virus infection, UFL1 is recruited to the membrane targeting protein 14-3-3epsilon. Further, I found that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling utilizing a series of RIG-I activation mutants. As I hypothesized that the primary role of UFL1 in RIG-I signaling regulation was through ufmylation of an interacting protein, I assessed the ufmylation status of 14-3-3epsilon, and I found that 14-3-3epsilon has an increase in covalent UFM1-conjugation when RIG-I activation is induced by viral infection. Additionally, loss of either cellular UFM1 or UFL1 expression prevents the interaction of 14-3-3epsilon with RIG-I. The consequences of which are impaired RIG-I recruitment to MAVS sites at ER-mitochondrial membranes. Indeed, I found that loss of UFM1 abrogates the interaction of RIG-I with MAVS via co-immunoprecipitation, and MAVS activation by oligomerization using semi-denaturing detergent agarose gel electrophoresis, and thus downstream signal transduction which induces IFN. These results reveal ufmylation as an integral regulatory component of the RIG-I signaling pathway and as a novel post-translational control for IFN induction. Having discovered a role for ufmylation in regulating the interaction of 14-3-3epsilon with RIG-I, I hypothesized that there might be other proteins regulated by ufmylation during RIG-I signaling, as PTMs play a crucial role in the regulation of a multitude of proteins in the antiviral response pathways. Utilizing an unbiased proteomics approach, I profiled all the UFM1-interacting proteins during viral infection. These data revealed multiple proteins that display differential UFM1-interaction, including TBK1, the serine-threonine kinase that plays a key role in the induction of IFNs. Follow-up work revealed that five of the top 8 proteins identified modulate IFN induction. Further studies to identify how ufmylation influences these proteins functions, and how their ufmylation status regulates their role in RIG-I signaling is needed, but this reveals a broad role for ufmylation as an important RIG-I pathway regulatory PTM. Further, as many intracellular pathogen sensing pathways share common signaling proteins, ufmylation may also regulate these signaling processes as well. Overall, my work has revealed a previously undiscovered node of RIG-I regulation and uncovered a new class of RIG-I regulatory proteins that become ufmylated during the antiviral response.
Item Open Access Successes and Challenges on the Road to Cure Hepatitis C.(PLoS Pathog, 2015-06) Horner, Stacy M; Naggie, Susanna