82 results on '"Stacy M. Horner"'
Search Results
2. The mRNA Cap 2′-O-Methyltransferase CMTR1 Regulates the Expression of Certain Interferon-Stimulated Genes
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Graham D. Williams, Nandan S. Gokhale, Daltry L. Snider, and Stacy M. Horner
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ISGs ,RNA modification ,antiviral ,interferon ,Microbiology ,QR1-502 - Abstract
ABSTRACT 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 are unknown. Here, we reveal that CMTR1, an ISG that catalyzes 2′-O-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 2′-O-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. IMPORTANCE Induction of an efficient type I IFN response is important to control viral infection. We show that the host 2′-O-methyltransferase CMTR1 facilitates the protein expression of ISGs in human cells by preventing IFIT1 from inhibiting the translation of those mRNAs lacking cap 2′-O-methylation. Thus, CMTR1 promotes the IFN-mediated antiviral response.
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- 2020
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3. A potentially abundant junctional RNA motif stabilized by m6A and Mg2+
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Bei Liu, Dawn K. Merriman, Seung H. Choi, Maria A. Schumacher, Raphael Plangger, Christoph Kreutz, Stacy M. Horner, Kate D. Meyer, and Hashim M. Al-Hashimi
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Science - Abstract
N 6-Methyladenosine (m6A) is a post-transcriptional RNA modification that modulates RNA structure through a destabilization of m6A base pairing. Here the authors use NMR and UV melting experiments and show that m6A can also stabilize m6A–U base pairs and global RNA structure when positioned adjacent to a 5ʹ bulge.
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- 2018
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4. A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time
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Michael J. McFadden, Aaron Mitchell-Dick, Christine Vazquez, Allison E. Roder, Kevin F. Labagnara, John J. McMahon, Debra L. Silver, and Stacy M. Horner
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Zika virus (ZIKV) ,NS2B-NS3 ,NS4B-NS5 ,reporter ,fluorescence ,live cell imaging ,apoptosis ,Microbiology ,QR1-502 - Abstract
Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.
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- 2018
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5. N6 -methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle
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Hasan Imam, Mohsin Khan, Nandan S. Gokhale, Alexa B. R. McIntyre, Geon-Woo Kim, Jae Young Jang, Seong-Jun Kim, Christopher E. Mason, Stacy M. Horner, and Aleem Siddiqui
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- 2018
- Full Text
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6. The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein.
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Allison E Roder, Christine Vazquez, and Stacy M Horner
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and 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. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. 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 wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV 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.
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- 2019
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7. Structure-first identification of RNA elements that regulate dengue virus genome architecture and replication
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Mark A. Boerneke, Nandan S. Gokhale, Stacy M. Horner, and Kevin M. Weeks
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Multidisciplinary - Abstract
The genomes of RNA viruses encode the information required for replication in host cells both in their linear sequence and in complex higher-order structures. A subset of these RNA genome structures show clear sequence conservation, and have been extensively described for well-characterized viruses. However, the extent to which viral RNA genomes contain functional structural elements—unable to be detected by sequence alone—that nonetheless are critical to viral fitness is largely unknown. Here, we devise a structure-first experimental strategy and use it to identify 22 structure-similar motifs across the coding sequences of the RNA genomes for the four dengue virus serotypes. At least 10 of these motifs modulate viral fitness, revealing a significant unnoticed extent of RNA structure-mediated regulation within viral coding sequences. These viral RNA structures promote a compact global genome architecture, interact with proteins, and regulate the viral replication cycle. These motifs are also thus 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 enables efficient discovery of pervasive RNA-mediated regulation in viral genomes and, likely, other cellular RNAs.
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- 2023
8. Structure-first identification of conserved RNA elements that regulate dengue virus genome architecture and replication
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Mark A. Boerneke, Nandan S. Gokhale, Stacy M. Horner, and Kevin M. Weeks
- Abstract
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.
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- 2022
9. N6-Methyladenosine Regulates Host Responses to Viral Infection
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Michael J. McFadden and Stacy M. Horner
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0303 health sciences ,Host (biology) ,viruses ,medicine.medical_treatment ,Biology ,Biochemistry ,Viral infection ,Cell function ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Immune system ,Cytokine ,chemistry ,RNA modification ,medicine ,Viral rna ,N6-Methyladenosine ,Molecular Biology ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Recent discoveries have revealed that, during viral infection, the presence of the RNA modification N6-methyladenosine (m6A) on viral and cellular RNAs has profound impacts on infection outcome. Although m6A directly regulates many viral RNA processes, its effects on cellular RNAs and pathways during infection have only recently begun to be elucidated. Disentangling the effects of m6A on viral and host RNAs remains a challenge for the field. m6A has been found to regulate host responses such as viral RNA sensing, cytokine responses, and immune cell functions. We highlight recent findings describing how m6A modulates host responses to viral infection and discuss future directions that will lead to a synergistic understanding of the processes by which m6A regulates viral infection.
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- 2021
10. WTAP targets the METTL3 m
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Matthew T, Sacco, Katherine M, Bland, and Stacy M, Horner
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Cell Nucleus ,Humans ,RNA, Viral ,Cell Cycle Proteins ,Hepacivirus ,Methyltransferases ,RNA Splicing Factors ,RNA, Messenger ,Hepatitis C ,Article ,Virus-Cell Interactions - Abstract
Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m(6)A) regulates the viral life cycle. This life cycle takes place solely in the cytoplasm, while m(6)A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m(6)A is deposited on the viral RNA have been unclear. In this work, we find that m(6)A modification of HCV RNA by the m(6)A-methyltransferase proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms’ tumor 1-associating protein (WTAP). WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m(6)A-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 m(6)A 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 m(6)A. Excitingly, WTAP regulation of both HCV RNA m(6)A modification and virion production was independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m(6)A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m(6)A 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 m(6)A modification with the cytoplasmic HCV life cycle. In this study, we present evidence for how m(6)A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m(6)A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m(6)A modification of cytoplasmic RNA viruses is likely relevant for other m(6)A-modified positive-strand RNA viruses with cytoplasmic life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.
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- 2022
11. WTAP targets the METTL3 m6A-methyltransferase complex to cytoplasmic hepatitis C virus RNA to regulate infection
- Author
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Matthew T. Sacco, Katherine M. Bland, and Stacy M. Horner
- Abstract
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.
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- 2022
12. Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing
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Jonathan S. Abebe, Alexa B. R. McIntyre, Matthew D. Weitzman, Alexander M. Price, Nandan S. Gokhale, Christopher E. Mason, Daniel P. Depledge, Ashley N. Della Fera, Katharina E. Hayer, Stacy M. Horner, and Angus C. Wilson
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0301 basic medicine ,Sequence analysis ,Science ,viruses ,genetic processes ,General Physics and Astronomy ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Transcriptome ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Gene Knockdown Techniques ,natural sciences ,lcsh:Science ,Multidisciplinary ,RNA ,DNA virus ,General Chemistry ,030104 developmental biology ,Viral replication ,chemistry ,RNA splicing ,lcsh:Q ,030217 neurology & neurosurgery ,DNA - Abstract
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, here we profile 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.
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- 2020
13. Signaling from the RNA sensor RIG-I is regulated by ufmylation
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Dia C. Beachboard, Stacy M. Horner, Moonhee Park, Daltry L. Snider, and Kristen A. Murphy
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Ubiquitin-Protein Ligases ,viruses ,RNA-binding protein ,chemical and pharmacologic phenomena ,RNA Virus Infections ,Humans ,Receptors, Immunologic ,Innate immune system ,Multidisciplinary ,biology ,RIG-I ,Chemistry ,Endoplasmic reticulum ,virus diseases ,RNA ,Signal transducing adaptor protein ,biochemical phenomena, metabolism, and nutrition ,Immunity, Innate ,Ubiquitin ligase ,Cell biology ,14-3-3 Proteins ,biology.protein ,DEAD Box Protein 58 ,RNA, Viral ,Interferons ,Signal transduction ,Signal Transduction - Abstract
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 14-3-3ε, and that this complex is then recruited to activated RIG-I to promote downstream innate immune signaling. Importantly, we found that 14-3-3ε has an increase in UFM1-conjugation following RIG-I activation. Additionally, loss of cellular ufmylation prevents the interaction of 14-3-3ε 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 14-3-3ε. As such, ufmylation positively regulates RIG-I recruitment to its signaling adaptor proteins MAVS for induction of interferon in response to RNA virus infection.
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- 2022
14. How RNA modifications regulate the antiviral response
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Matthew T. Sacco, Matthew G Thompson, and Stacy M. Horner
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Messenger RNA ,Adenosine ,Immunology ,Pattern recognition receptor ,Cellular homeostasis ,RNA ,Biology ,Antiviral Agents ,Article ,Immunity, Innate ,Cell biology ,chemistry.chemical_compound ,chemistry ,RNA editing ,Virus Diseases ,Gene expression ,Nucleic acid ,Immunology and Allergy ,Humans ,RNA, Viral ,N6-Methyladenosine - Abstract
Induction of the antiviral innate immune response is highly regulated at the RNA level, particularly by RNA modifications. Recent discoveries have revealed how RNA modifications play key roles in cellular surveillance of nucleic acids and in controlling gene expression in response to viral infection. These modifications have emerged as being essential for a functional antiviral response and maintaining cellular homeostasis. In this review, we will highlight these and other discoveries that describe how the antiviral response is controlled by modifications to both viral and cellular RNA, focusing on how mRNA cap modifications, N6-methyladenosine, and RNA editing all contribute to coordinating an efficient response that properly controls viral infection.
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- 2021
15. Proteomic analysis of mitochondrial-associated ER membranes (MAM) during RNA virus infection reveals dynamic changes in protein and organelle trafficking.
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Stacy M Horner, Courtney Wilkins, Samantha Badil, Jason Iskarpatyoti, and Michael Gale
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Medicine ,Science - Abstract
RIG-I pathway signaling of innate immunity against RNA virus infection is organized between the ER and mitochondria on a subdomain of the ER called the mitochondrial-associated ER membrane (MAM). The RIG-I adaptor protein MAVS transmits downstream signaling of antiviral immunity, with signaling complexes assembling on the MAM in association with mitochondria and peroxisomes. To identify components that regulate MAVS signalosome assembly on the MAM, we characterized the proteome of MAM, ER, and cytosol from cells infected with either chronic (hepatitis C) or acute (Sendai) RNA virus infections, as well as mock-infected cells. Comparative analysis of protein trafficking dynamics during both chronic and acute viral infection reveals differential protein profiles in the MAM during RIG-I pathway activation. We identified proteins and biochemical pathways recruited into and out of the MAM in both chronic and acute RNA viral infections, representing proteins that drive immunity and/or regulate viral replication. In addition, by using this comparative proteomics approach, we identified 3 new MAVS-interacting proteins, RAB1B, VTN, and LONP1, and defined LONP1 as a positive regulator of the RIG-I pathway. Our proteomic analysis also reveals a dynamic cross-talk between subcellular compartments during both acute and chronic RNA virus infection, and demonstrates the importance of the MAM as a central platform that coordinates innate immune signaling to initiate immunity against RNA virus infection.
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- 2015
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16. The m 6 A reader IMP2 directs autoimmune inflammation through an IL-17– and TNFα-dependent C/EBP transcription factor axis
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Nandan S. Gokhale, Sarah L. Gaffen, Rami Bechara, Nilesh Amatya, Stacy M. Horner, Yang Li, Rachel D. Bailey, Bianca M. Coleman, Partha S. Biswas, Felix E. Y. Aggor, Chetan V. Jawale, Ning Dai, Anita Bansal, Tiffany C. Taylor, Amanda C. Poholek, and De-Dong Li
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0301 basic medicine ,Messenger RNA ,Chemistry ,Effector ,MRNA modification ,Immunology ,General Medicine ,MRNA stabilization ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Enhancer binding ,CEBPB ,Tumor necrosis factor alpha ,Transcription factor - Abstract
Excessive cytokine activity underlies many autoimmune conditions, particularly through the interleukin-17 (IL-17) and tumor necrosis factor-α (TNFα) signaling axis. Both cytokines activate nuclear factor κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably, CCAAT/enhancer binding proteins (C/EBPs). Here, we demonstrate the unexpected involvement of a posttranscriptional "epitranscriptomic" mRNA modification [N6-methyladenosine (m6A)] in regulating C/EBPβ and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPβ/δ-encoding transcripts contain m6A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m6A modification. Induction of C/EBPs is enhanced by an m6A methylase "writer" and suppressed by a demethylase "eraser." The only m6A "reader" found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m6A modification. IMP2 facilitated IL-17-mediated Cebpd mRNA stabilization and promoted translation of C/EBPβ/δ in response to IL-17A, IL-17F, and TNFα. RNA sequencing revealed transcriptome-wide IL-17-induced transcripts that are IMP2 influenced, and RNA immunoprecipitation sequencing identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2, and C/EBPβ/δ. Imp2-/- mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2 Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, posttranscriptional regulation of C/EBPs through m6A/IMP2 represents a previously unidentified paradigm of cytokine-driven autoimmune inflammation.
- Published
- 2021
17. FTO suppresses STAT3 activation and modulates proinflammatory interferon-stimulated gene expression
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Stacy M. Horner, Kim Y. Somfleth, Matthew T. Sacco, Nandan S. Gokhale, Moonhee Park, Kristen A. Murphy, and Michael J. McFadden
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STAT3 Transcription Factor ,Alpha-Ketoglutarate-Dependent Dioxygenase FTO ,Gene Expression ,Article ,Proinflammatory cytokine ,Structural Biology ,Interferon ,Transcription (biology) ,medicine ,Humans ,RNA, Messenger ,STAT3 ,Molecular Biology ,Transcription factor ,Inflammation ,Messenger RNA ,biology ,Chemistry ,Interferon-stimulated gene ,MRNA modification ,nutritional and metabolic diseases ,Methyltransferases ,Cell biology ,Gene Expression Regulation ,Interferon Type I ,biology.protein ,Demethylase ,medicine.drug - Abstract
Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase FTO in the type I IFN response. FTO, which can remove either m6A or the cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation is not through the actions of FTO on m6Am. Further, we found that depletion of FTO led to activation of STAT3, a transcription factor that mediates responses to various cytokines, but whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.
- Published
- 2021
18. Regulation of Viral Infection by the RNA Modification N6-Methyladenosine
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Nandan S. Gokhale, Graham D. Williams, and Stacy M. Horner
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0303 health sciences ,Innate immune system ,Regulator ,RNA ,Biology ,Viral infection ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Immune system ,chemistry ,Virology ,RNA modification ,N6-Methyladenosine ,Post-transcriptional regulation ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
In recent years, the RNA modification N6-methyladenosine (m6A) has been found to play a role in the life cycles of numerous viruses and also in the cellular response to viral infection. m6A has emerged as a regulator of many fundamental aspects of RNA biology. Here, we highlight recent advances in techniques for the study of m6A, as well as advances in our understanding of the cellular machinery that controls the addition, removal, recognition, and functions of m6A. We then summarize the many newly discovered roles of m6A during viral infection, including how it regulates innate and adaptive immune responses to infection. Overall, the goals of this review are to summarize the roles of m6A on both cellular and viral RNAs and to describe future directions for uncovering new functions of m6A during infection.
- Published
- 2019
19. The small GTPase RAB1B promotes antiviral innate immunity by interacting with TNF receptor–associated factor 3 (TRAF3)
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Dia C. Beachboard, Daltry L. Snider, Madhuvanthi Vijayan, Michael J. McFadden, Sydney Stanley, Stacy M. Horner, Dillon J. Fernando, Graham D. Williams, and Moonhee Park
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0301 basic medicine ,TRAF3 ,Immunology ,Biology ,Biochemistry ,03 medical and health sciences ,Interferon ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Receptors, Immunologic ,Vero Cells ,Molecular Biology ,Mitochondrial antiviral-signaling protein ,Innate immune system ,TNF Receptor-Associated Factor 3 ,030102 biochemistry & molecular biology ,Zika Virus Infection ,Pattern recognition receptor ,Signal transducing adaptor protein ,Interferon-beta ,Cell Biology ,Immunity, Innate ,Cell biology ,rab1 GTP-Binding Proteins ,HEK293 Cells ,030104 developmental biology ,DEAD Box Protein 58 ,IRF3 ,Protein Binding ,Signal Transduction ,medicine.drug - Abstract
Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor–associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-β induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I–mediated signaling to IFN-β and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.
- Published
- 2019
20. N
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Michael J, McFadden and Stacy M, Horner
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Adenosine ,Virus Diseases ,viruses ,Cytokines ,Humans ,RNA, Viral ,Immunity, Innate ,Article - Abstract
Recent discoveries have revealed that during viral infection the presence of the RNA modification N6-methyladenosine (m(6)A) on viral and cellular RNAs has profound impacts on infection outcome. While m(6)A directly regulates many viral RNA processes, its effects on cellular RNAs and pathways during infection have only recently begun to be elucidated. Disentangling the effects of m(6)A on viral and host RNAs remains a challenge for the field. m(6)A has been found to regulate host responses such as viral RNA sensing, cytokine responses, and immune cell functions. We highlight recent findings describing how m(6)A modulates host responses to viral infection and discuss future directions that will lead to a synergistic understanding of the processes by which m(6)A regulates viral infection.
- Published
- 2020
21. Flipping the script: viral capitalization of RNA modifications
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Stacy M. Horner and Matthew T. Sacco
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Adenosine ,viruses ,Computational biology ,Biology ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Viral life cycle ,Genetics ,RNA, Messenger ,Molecular Biology ,Gene ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Review Paper ,2'-O-methylation ,RNA ,RNA-Binding Proteins ,Translation (biology) ,General Medicine ,chemistry ,RNA, Viral ,N6-Methyladenosine ,030217 neurology & neurosurgery ,Function (biology) - Abstract
RNA encoded by RNA viruses is highly regulated so that it can function in multiple roles during the viral life cycle. These roles include serving as the mRNA template for translation or the genetic material for replication as well as being packaged into progeny virions. RNA modifications provide an emerging regulatory dimension to the RNA of viruses. Modification of the viral RNA can increase the functional genomic capacity of the RNA viruses without the need to encode and translate additional genes. Further, RNA modifications can facilitate interactions with host or viral RNA-binding proteins that promote replication or can prevent interactions with antiviral RNA-binding proteins. The mechanisms by which RNA viruses facilitate modification of their RNA are diverse. In this review, we discuss some of these mechanisms, including exploring the unknown mechanism by which the RNA of viruses that replicate in the cytoplasm could acquire the RNA modification N6-methyladenosine.
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- 2020
22. Post-transcriptional regulation of antiviral gene expression by N6-methyladenosine
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Blerta Xhemalce, Michael J. McFadden, Nathan S. Abell, Haralambos Mourelatos, Stacy M. Horner, Christopher E. Mason, Alexa B. R. McIntyre, Nandan S. Gokhale, and Hélène Ipas
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0301 basic medicine ,Methyltransferase ,Adenosine ,Transcription, Genetic ,Biology ,Virus Replication ,Antiviral Agents ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Interferon ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Ribosome profiling ,RNA Processing, Post-Transcriptional ,Post-transcriptional regulation ,Gene ,Vero Cells ,Methyltransferase complex ,RNA ,virus diseases ,RNA-Binding Proteins ,Translation (biology) ,Interferon-beta ,Methyltransferases ,Vesiculovirus ,Antigens, Differentiation ,Cell biology ,030104 developmental biology ,HEK293 Cells ,chemistry ,A549 Cells ,Protein Biosynthesis ,Host-Pathogen Interactions ,N6-Methyladenosine ,Vesicular Stomatitis ,030217 neurology & neurosurgery ,medicine.drug - Abstract
SUMMARY Type I interferons (IFNs) induce hundreds of IFN-stimulated genes (ISGs) in response to viral infection. Induction of these ISGs must be regulated for an efficient and controlled antiviral response, but post-transcriptional of these genes have not been well defined. Here, we identify a 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 identify a subset of ISGs, including IFITM1, whose translation is enhanced by m6A and the m6A methyltransferase proteins METTL3 and METTL14. We further determine that the m6A reader YTHDF1 increases the expression of IFITM1 in an m6A-binding-dependent manner. Importantly, we find that the m6A methyltransferase complex promotes the antiviral activity of type I IFN. Thus, these studies identify m6A as having a role in post-transcriptional control of ISG translation during the type I IFN response for antiviral restriction., Graphical Abstract, In brief McFadden et al. report that the transcripts of many interferon-stimulated genes (ISGs), which encode antiviral proteins, are m6A-modified. m6A promotes the translation of certain ISGs, enhancing the antiviral effects of interferon. This study adds to our understanding of the functions of m6A at the virus-host interface.
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- 2020
23. The mRNA Cap 2′- O -Methyltransferase CMTR1 Regulates the Expression of Certain Interferon-Stimulated Genes
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Daltry L. Snider, Stacy M. Horner, Graham D. Williams, and Nandan S. Gokhale
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lcsh:QR1-502 ,ISGs ,Biology ,Microbiology ,lcsh:Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Interferon ,medicine ,Molecular Biology ,Gene ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Gene knockdown ,RNA ,Translation (biology) ,interferon ,RNA modification ,antiviral ,ISG15 ,QR1-502 ,3. Good health ,Cell biology ,Signal transduction ,030217 neurology & neurosurgery ,medicine.drug - Abstract
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 are unknown. Here, we reveal that CMTR1, an ISG that catalyzes 2′-O-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 2′-O-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. IMPORTANCE Induction of an efficient type I IFN response is important to control viral infection. We show that the host 2′-O-methyltransferase CMTR1 facilitates the protein expression of ISGs in human cells by preventing IFIT1 from inhibiting the translation of those mRNAs lacking cap 2′-O-methylation. Thus, CMTR1 promotes the IFN-mediated antiviral response.
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- 2020
24. The m
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Rami, Bechara, Nilesh, Amatya, Rachel D, Bailey, Yang, Li, Felix E Y, Aggor, De-Dong, Li, Chetan V, Jawale, Bianca M, Coleman, Ning, Dai, Nandan S, Gokhale, Tiffany C, Taylor, Stacy M, Horner, Amanda C, Poholek, Anita, Bansal, Partha S, Biswas, and Sarah L, Gaffen
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Inflammation ,Male ,Mice, Knockout ,Adenosine ,Tumor Necrosis Factor-alpha ,Interleukin-17 ,RNA-Binding Proteins ,Autoimmunity ,Article ,Cell Line ,Mice, Inbred C57BL ,CCAAT-Enhancer-Binding Proteins ,Animals ,Humans ,Female - Abstract
Excessive cytokine activity underlies many autoimmune conditions, particularly through the IL-17 and TNFα signaling axes. Both cytokines activate NF-κB, but appropriate induction of downstream effector genes requires coordinated activation of other transcription factors, notably CCAAT/Enhancer Binding Proteins (C/EBPs). Here we demonstrate the unexpected involvement of a post-transcriptional ‘epitranscriptomic’ mRNA modification (N6-methyladenosine, m(6)A) in regulating C/EBPβ and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPβ/δ-encoding transcripts contain m(6)A consensus sites, we show that Cebpd and Cebpb mRNAs are subject to m(6)A modification. Induction of C/EBPs is enhanced by an m(6)A methylase ‘writer’ and suppressed by a demethylase ‘eraser.’ The only m(6)A ‘reader’ found to be involved in this pathway was IGF2BP2 (IMP2), and IMP2 occupancy of Cebpd and Cebpb mRNA was enhanced by m(6)A modification. IMP2 facilitated IL-17-mediated Cebpd mRNA stabilization and promoted translation of C/EBPβ/δ in response to IL-17A, IL-17F and TNFα. RNASeq revealed transcriptome-wide IL-17-induced transcripts that are IMP2-influenced, and RIPSeq identified the subset of mRNAs that are directly occupied by IMP2, which included Cebpb and Cebpd. Lipocalin-2 (Lcn2), a hallmark of autoimmune kidney injury, was strongly dependent on IL-17, IMP2 and C/EBPβ/δ. Indeed, Imp2(−/−) mice were resistant to autoantibody-induced glomerulonephritis (AGN), showing impaired renal expression of C/EBPs and Lcn2. Moreover, IMP2 deletion initiated only after AGN onset ameliorated disease. Thus, post-transcriptional regulation of C/EBPs through m(6)A/IMP2 represents a new paradigm of cytokine-driven autoimmune inflammation.
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- 2020
25. Comprehensive Multi-omics Analysis Reveals Mitochondrial Stress as a Central Biological Hub for Spaceflight Impact
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Kathleen M. Fisch, Sara Brin Rosenthal, Yared H. Kidane, Jeffrey S. Willey, Benjamin Stear, Yue Ying, Sara R. Zwart, Robert Meller, Stacy M. Horner, Sylvain V. Costes, Man S. Kim, Nicolae Sapoval, Evagelia C. Laiakis, Komal S. Rathi, R. A. Leo Elworth, Susana Zanello, Adrienne Nugent, Helio A. Costa, Yuanchao Zhang, Dong Wang, Afshin Beheshti, Larry N. Singh, Willian A. da Silveira, Todd J. Treangen, Matthew MacKay, Nandan S. Gokhale, Christopher E. Mason, Hossein Fazelinia, Sonja Schrepfer, Douglas C. Wallace, Jonathan C. Schisler, Cem Meydan, Jonathan Foox, Gary Hardiman, J. Tyson McDonald, Brian Crucian, Scott M. Smith, and Deanne Taylor
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DNA damage ,Systems biology ,Computational biology ,Mitochondrion ,Biology ,Spaceflight ,General Biochemistry, Genetics and Molecular Biology ,Article ,law.invention ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Metabolomics ,law ,Stress, Physiological ,Animals ,Humans ,Epigenetics ,030304 developmental biology ,0303 health sciences ,Mice, Inbred BALB C ,Muscles ,Genomics ,Space Flight ,Lipid Metabolism ,Phenotype ,Immunity, Innate ,Metabolic Flux Analysis ,Circadian Rhythm ,Extracellular Matrix ,Mitochondria ,Mice, Inbred C57BL ,Smell ,Organ Specificity ,030217 neurology & neurosurgery - Abstract
Spaceflight is known to impose changes on human physiology with unknown molecular etiologies. To reveal these causes, we used a multi-omics, systems biology analytical approach using biomedical profiles from fifty-nine astronauts and data from NASA's GeneLab derived from hundreds of samples flown in space to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight. Overall pathway analyses on the multi-omics datasets showed significant enrichment for mitochondrial processes, as well as innate immunity, chronic inflammation, cell cycle, circadian rhythm, and olfactory functions. Importantly, NASA's Twin Study provided a platform to confirm several of our principal findings. Evidence of altered mitochondrial function and DNA damage was also found in the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight.
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- 2020
26. Shotgun Transcriptome and Isothermal Profiling of SARS-CoV-2 Infection Reveals Unique Host Responses, Viral Diversification, and Drug Interactions
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David Danko, Ari Melnick, Fritz J. Sedlazeck, Matthew MacKay, Melissa M. Cushing, Lin Cong, Robert E. Schwartz, Massimo Loda, Lars F. Westblade, Daniela Bezdan, Jonathan Foox, Diana Pohle, Peter A D Steel, Craig Westover, Nicholas P. Tatonetti, John Sipley, Arryn Craney, Amos J Shemesh, Danielle Thierry-Mieg, Iman Hajirasouliha, Shawn Levy, Alon Shaiber, Daniel Butler, Vijendra Ramlall, Undina Gisladottir, Krista Ryon, Dong Xu, Chandrima Bhattacharya, Michael Zietz, Joel Rosiene, Shixiu Wu, Hanna Rennert, Jenny Xiang, Maria A. Sierra, Nikolay A. Ivanov, Bradley W. Langhorst, Nathan A. Tanner, P. Ruggiero, Mirella Salvatore, Priya Velu, Justyna Gawrys, Cem Meydan, Benjamin Young, Ebrahim Afshinnekoo, Stacy M. Horner, Dmitry Meleshko, Christopher Mozsary, Thomas Iftner, Angelika Iftner, Christopher E. Mason, Jean Thierry-Mieg, and Marcin Imielinski
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Drug ,0303 health sciences ,media_common.quotation_subject ,Outbreak ,Diseases ,Subclade ,Shotgun ,RNA-Seq ,Biology ,Virology ,Article ,Computational biology and bioinformatics ,3. Good health ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Interferon ,Pandemic ,medicine ,030217 neurology & neurosurgery ,030304 developmental biology ,medicine.drug ,media_common - Abstract
In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin–angiotensin–aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies., Here, using clinical samples and autopsy tissues, the authors combine fast-colorimetric test (LAMP) for SARS-CoV-2 infection and large-scale shotgun metatranscriptomics for host, viral, and microbial profiling and provide a map of the viral genetic features of the New York City outbreak and associate specific host responses and gene expression perturbations with SARS-CoV-2 infection.
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- 2020
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27. Limits in the detection of m6A changes using MeRIP/m6A-seq
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Alexa B. R. McIntyre, Leandro Cerchietti, Stacy M. Horner, Nandan S. Gokhale, Samie R. Jaffrey, and Christopher E. Mason
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Standards ,Adenosine ,Statistical methods ,Sequence analysis ,Immunoprecipitation ,lcsh:Medicine ,Computational biology ,Common method ,Methylation ,Article ,03 medical and health sciences ,0302 clinical medicine ,Gene expression ,Humans ,Base sequence ,RNA, Messenger ,lcsh:Science ,skin and connective tissue diseases ,030304 developmental biology ,0303 health sciences ,Reproducibility ,Multidisciplinary ,Base Sequence ,Chemistry ,Sequence Analysis, RNA ,lcsh:R ,RNA ,RNA sequencing ,Replicate ,RNA modification ,Rna immunoprecipitation ,lcsh:Q ,sense organs ,030217 neurology & neurosurgery ,Software ,Algorithms - Abstract
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.
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- 2020
28. The mRNA cap 2’O methyltransferase CMTR1 regulates the expression of certain interferon-stimulated genes
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Graham D. Williams, Stacy M. Horner, Nandan S. Gokhale, and Daltry L. Snider
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0303 health sciences ,Messenger RNA ,Gene knockdown ,030302 biochemistry & molecular biology ,RNA ,Methylation ,Biology ,ISG15 ,Cell biology ,03 medical and health sciences ,Interferon ,medicine ,Signal transduction ,Gene ,030304 developmental biology ,medicine.drug - Abstract
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 2’O 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 2’O 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 2’O methyltransferase CMTR1 facilitates the protein expression of ISGs in human cells by preventing IFIT1 from inhibiting the translation of these mRNAs lacking cap 2’O methylation. Thus, CMTR1 promotes the IFN-mediated antiviral response.
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- 2020
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29. Temporal Telomere and DNA Damage Responses in the Space Radiation Environment
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Stacy M. Horner, Miles J. McKenna, Kumar Sharma, Daniela Bezdan, Scott M. Smith, Viktor R. Drel, Nandan S. Gokhale, Susan M. Bailey, Jonathan Foox, Sara R. Zwart, Jared J. Luxton, Cem Meydan, Kirill Grigorev, Daniel Butler, Kerry George, Brian Crucian, Christopher E. Mason, and Lynn Taylor
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Chronic exposure ,Biomarker ,Lifestyle factors ,Cell division ,DNA damage ,law ,Biology ,Spaceflight ,Space radiation ,Telomere ,Cell biology ,law.invention - Abstract
We assessed telomere length dynamics in three unrelated astronauts before, during, and after 1-year or 6-month missions aboard the International Space Station. Telomeres, repetitive terminal features of chromosomes essential for maintaining genome integrity, shorten with cell division, lifestyle factors and stresses, and environmental exposures, and so provide a robust biomarker of health, aging, and age-related diseases. Similar to our results for NASA’s One-Year Mission twin astronaut (Garrett-Bakelman et al., 2019), significantly longer telomeres were observed during spaceflight for two 6-month mission astronauts. Furthermore, telomere length shortened rapidly after return to Earth for all three crewmembers and, overall, telomere length tended to be shorter after spaceflight than before. Consistent with chronic exposure to the space radiation environment, signatures of persistent DNA damage responses were also detected, which included mitochondrial and oxidative stress, as well as telomeric and chromosomal aberrations, and together provide potential mechanistic insight into spaceflight-specific telomere elongation.
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- 2020
30. Convergent evolution of escape from hepaciviral antagonism in primates.
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Maulik R Patel, Yueh-Ming Loo, Stacy M Horner, Michael Gale, and Harmit S Malik
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Biology (General) ,QH301-705.5 - Abstract
The ability to mount an interferon response on sensing viral infection is a critical component of mammalian innate immunity. Several viruses directly antagonize viral sensing pathways to block activation of the host immune response. Here, we show that recurrent viral antagonism has shaped the evolution of the host protein MAVS--a crucial component of the viral-sensing pathway in primates. From sequencing and phylogenetic analyses of MAVS from 21 simian primates, we found that MAVS has evolved under strong positive selection. We focused on how this positive selection has shaped MAVS' susceptibility to Hepatitis C virus (HCV). We functionally tested MAVS proteins from diverse primate species for their ability to resist antagonism by HCV, which uses its protease NS3/4A to cleave human MAVS. We found that MAVS from multiple primates are resistant to inhibition by the HCV protease. This resistance maps to single changes within the protease cleavage site in MAVS, which protect MAVS from getting cleaved by the HCV protease. Remarkably, most of these changes have been independently acquired at a single residue 506 that evolved under positive selection. We show that "escape" mutations lower affinity of the NS3 protease for MAVS and allow it to better restrict HCV replication. We further show that NS3 proteases from all other primate hepaciviruses, including the highly divergent GBV-A and GBV-C viruses, are functionally similar to HCV. We conclude that convergent evolution at residue 506 in multiple primates has resulted in escape from antagonism by hepaciviruses. Our study provides a model whereby insights into the ancient history of viral infections in primates can be gained using extant host and virus genes. Our analyses also provide a means by which primates might clear infections by extant hepaciviruses like HCV.
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- 2012
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31. Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing
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Matthew D. Weitzman, Alexa B. R. McIntyre, Angus C. Wilson, Katharina E. Hayer, Ashley N. Della Fera, Alexander M. Price, Nandan S. Gokhale, Stacy M. Horner, Christopher E. Mason, and Daniel P. Depledge
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chemistry.chemical_classification ,Transcriptome ,Rna processing ,chemistry ,RNA splicing ,RNA ,Nucleotide ,DNA virus ,Computational biology ,Biology ,Pathogen ,Function (biology) - Abstract
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.
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- 2019
32. N6 -methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle
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Stacy M. Horner, Hasan Imam, Alexa B. R. McIntyre, Aleem Siddiqui, Jae Young Jang, Geon-Woo Kim, Nandan S. Gokhale, Mohsin Khan, Christopher E. Mason, and Seong-Jun Kim
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0301 basic medicine ,Hepatitis B virus ,Adenosine ,RNA methylation ,RNA Stability ,Chronic Liver Disease and Cirrhosis ,Biology ,medicine.disease_cause ,Hepatitis ,Hepatitis - B ,Viral Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Gene expression ,Genetics ,medicine ,Humans ,Gene silencing ,Viral ,HBV reverse transcription ,epsilon loop ,Multidisciplinary ,Liver Disease ,RNA ,Reverse Transcription ,Hep G2 Cells ,Biological Sciences ,Stem Cell Research ,Stem-loop ,Reverse transcriptase ,Cell biology ,Infectious Diseases ,Good Health and Well Being ,030104 developmental biology ,Gene Expression Regulation ,chemistry ,030220 oncology & carcinogenesis ,Nucleic Acid Conformation ,N6-Methyladenosine ,Digestive Diseases ,Infection - Abstract
N6-methyladenosine (m(6)A) RNA methylation is the most abundant epitranscriptomic modification of eukaryotic messenger RNAs (mRNAs). Previous reports have found m(6)A on both cellular and viral transcripts and defined its role in regulating numerous biological processes, including viral infection. Here, we show that m(6)A and its associated machinery regulate the life cycle of hepatitis B virus (HBV). HBV is a DNA virus that completes its life cycle via an RNA intermediate, termed pregenomic RNA (pgRNA). Silencing of enzymes that catalyze the addition of m(6)A to RNA resulted in increased HBV protein expression, but overall reduced reverse transcription of the pgRNA. We mapped the m(6)A site in the HBV RNA and found that a conserved m(6)A consensus motif situated within the epsilon stem loop structure, is the site for m(6)A modification. The epsilon stem loop is located in the 3′ terminus of all HBV mRNAs and at both the 5′ and 3′ termini of the pgRNA. Mutational analysis of the identified m(6)A site in the 5′ epsilon stem loop of pgRNA revealed that m(6)A at this site is required for efficient reverse transcription of pgRNA, while m(6)A methylation of the 3′ epsilon stem loop results in destabilization of all HBV transcripts, suggesting that m(6)A has dual regulatory function for HBV RNA. Overall, this study reveals molecular insights into how m(6)A regulates HBV gene expression and reverse transcription, leading to an increased level of understanding of the HBV life cycle.
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- 2018
33. RNA modification of an RNA modifier prevents self-RNA sensing
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Stacy M. Horner and Daltry L. Snider
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Messenger RNA ,General Immunology and Microbiology ,QH301-705.5 ,General Neuroscience ,RNA ,MDA5 ,Translation (biology) ,Biology ,Adenosine ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Adenosine deaminase ,Interferon ,medicine ,biology.protein ,Biology (General) ,Nucleic acid structure ,General Agricultural and Biological Sciences ,medicine.drug - Abstract
A new study in PLOS Biology finds that interferon (IFN)-induced adenosine deaminase acting on RNA 1 (ADAR1) mRNA is N6-methyladenosine (m6A) modified to promote its translation, enabling ADAR1 to modify self-double-stranded RNAs (dsRNAs) generated during the IFN response and preventing activation of the melanoma differentiation-associated protein 5 (MDA5)-mediated host antiviral response.
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- 2021
34. RNA m6A methylation guides IL-17-driven autoimmunity through IMP2-dependent regulation of C/EBP transcription factors
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Rami Bechara, Nilesh Amatya, Rachel D. Bailey, Yang Li, Felix EY Aggor, De-Dong Li, Chetan V. Jawale, Bianca M. Coleman, Ning Dai, Nandan S. Gokhale, Tiffany C. Taylor, Stacy M. Horner, Amanda C. Poholek, Partha S. Biswas, and Sarah L. Gaffen
- Subjects
Immunology ,Immunology and Allergy - Abstract
Dysregulated activity of IL-17 underlies many autoimmune conditions, but the molecular mechanisms by which IL-17 mediates pathogenic inflammation remain poorly understood. IL-17 regulates pathogenic inflammatory genes by two key transcription factor classes, NF-κB and CCAAT/Enhancer Binding (C/EBP) proteins. Surprisingly little is known about mechanisms that activate C/EBPs. In seeking to understand how IL-17 upregulates C/EBPs, we found that IL-17 signaling enhanced Cebpd mRNA stability, concomitant with increased levels of C/EBPδ translation. In contrast, IL-17 had only a marginal inductive effect on Cebpb mRNA, yet C/EBPβ protein was strongly upregulated. Examination of Cebpb and Cebpd noncoding sequences identified consensus sites for N6-methyladenosine (m6A) modification, an epitranscriptomic mark that influences mRNA fate. knockdown of the m6A ‘writer’ METTL3 decreased C/EBP expression, which was reversed by the ‘eraser’ FTO. Moreover, we found that loss of an unusual m6A ‘reader’ IGF2BP2 (IMP2), an RNA binding protein known to control mRNA stability, impaired IL-17 induction of C/EBPs. IMP2 bound directly to Cebps transcripts, leading to enhanced Cebpd half-life and enhanced translation of both C/EBPs. Transcriptomic analysis revealed that IMP2 regulates C/EBP-dependent genes, including IL-6 and Lcn2. Lcn2 is a biomarker of autoantibody-induced glomerulonephritis (AGN), a setting of IL-17-driven inflammatory nephritis. Imp2−/− mice were resistant to AGN, which was linked to impaired upregulation of C/EPBs and Lcn2 in kidney. Thus, IL-17-induced autoimmunity is mediated through m6A-dependent post-transcriptional regulation of C/EBP transcription factors.
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- 2021
35. Regulation of Viral Infection by the RNA Modification
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Graham D, Williams, Nandan S, Gokhale, and Stacy M, Horner
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Mice ,Adenosine ,Gene Expression Regulation ,Host Microbial Interactions ,Virus Diseases ,Animals ,Humans ,RNA, Viral ,Adaptive Immunity ,RNA Processing, Post-Transcriptional ,Virus Replication ,Immunity, Innate ,Article - Abstract
In recent years, the RNA modification N6-methyladenosine (m(6)A) has been found to play a role in the life cycles of numerous viruses and also in the cellular response to viral infection. m(6)A has emerged as a regulator of many fundamental aspects of RNA biology. Here, we highlight recent advances in techniques for the study of m(6)A, as well as advances in our understanding of the cellular machinery that controls the addition, removal, recognition, and functions of m(6)A. We then summarize the many newly discovered roles of m(6)A during viral infection, including how it regulates innate and adaptive immune responses to infection. Overall, the goals of this review are to summarize these roles of m(6)A on both cellular and viral RNAs and to describe future directions for uncovering new functions of m(6)A during infection.
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- 2019
36. Altered m
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Nandan S, Gokhale, Alexa B R, McIntyre, Melissa D, Mattocks, Christopher L, Holley, Helen M, Lazear, Christopher E, Mason, and Stacy M, Horner
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Adenosine ,Zika Virus Infection ,viruses ,Flaviviridae ,RNA-Binding Proteins ,Hepacivirus ,Zika Virus ,Dengue Virus ,Flaviviridae Infections ,Protein Serine-Threonine Kinases ,Virus Replication ,Hepatitis C ,Article ,Cell Line ,Dengue ,Host-Pathogen Interactions ,Humans ,RNA, Messenger - Abstract
The RNA modification N6-methyladenosine (m(6)A) modulates mRNA fate and thus affects many biological processes. We analyzed m(6)A 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 m(6)A modification of specific cellular transcripts, including RIOK3 and CIRBP. During viral infection, the addition of m(6)A to RIOK3 promotes its translation, while loss of m(6)A in CIRBP promotes alternative splicing. Importantly, viral activation of innate immune sensing or the endoplasmic reticulum (ER) stress response contributes to the changes in m(6)A in RIOK3 and CIRBP, respectively. Further, several transcripts with infection-altered m(6)A 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 m(6)A modification of host mRNAs to regulate infection.
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- 2019
37. IL-27 signaling activates skin cells to induce innate antiviral proteins and protects against Zika virus infection
- Author
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Chelsea Handfield, Helen M. Lazear, Stacy M. Horner, Michael J. McFadden, Jutamas Suwanpradid, Mrinal K. Sarkar, Amanda S. MacLeod, Jessica Shannon, Peter Hoang, Lauren Pontius Floyd, J. Kwock, Ranjitha Uppala, David L. Corcoran, Johann E. Gudjonsson, Kevin F. Labagnara, and Gregory D. Sempowski
- Subjects
Functional role ,Keratinocytes ,First line ,Immunology ,Antiviral protein ,Gene Expression ,Biology ,Zika virus ,Cell Line ,03 medical and health sciences ,Antiviral immunity ,0302 clinical medicine ,Immune system ,Virology ,Humans ,STAT1 ,Research Articles ,Cells, Cultured ,030304 developmental biology ,Disease Resistance ,Skin ,0303 health sciences ,Multidisciplinary ,integumentary system ,Zika Virus Infection ,Interleukins ,SciAdv r-articles ,Zika Virus ,biology.organism_classification ,Phenotype ,Immunity, Innate ,3. Good health ,STAT1 Transcription Factor ,030220 oncology & carcinogenesis ,Host-Pathogen Interactions ,biology.protein ,Cytokines ,Biomarkers ,Research Article ,Signal Transduction - Abstract
IL-27 signaling activates the production of antiviral proteins in the skin and inhibits Zika virus infection., In the skin, antiviral proteins and other immune molecules serve as the first line of innate antiviral defense. Here, we identify and characterize the induction of cutaneous innate antiviral proteins in response to IL-27 and its functional role during cutaneous defense against Zika virus infection. Transcriptional and phenotypic profiling of epidermal keratinocytes treated with IL-27 demonstrated activation of antiviral proteins OAS1, OAS2, OASL, and MX1 in the skin of both mice and humans. IL-27–mediated antiviral protein induction was found to occur in a STAT1- and IRF3-dependent but STAT2-independent manner. Moreover, using IL27ra mice, we demonstrate a significant role for IL-27 in inhibiting Zika virus morbidity and mortality following cutaneous, but not intravenous, inoculation. Together, our results demonstrate a critical and previously unrecognized role for IL-27 in cutaneous innate antiviral immunity against Zika virus.
- Published
- 2019
38. Hepatitis C virus infection is inhibited by a non-canonical antiviral signaling pathway targeted by NS3-NS4A
- Author
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Stacy M. Horner, Chin Yee Tan, and Christine Vazquez
- Subjects
Interferon-Induced Helicase, IFIH1 ,viruses ,Immunology ,Hepacivirus ,Protein Serine-Threonine Kinases ,Viral Nonstructural Proteins ,Virus Replication ,Antiviral Agents ,Microbiology ,Gene Knockout Techniques ,03 medical and health sciences ,0302 clinical medicine ,Interferon ,Cell Line, Tumor ,Virology ,medicine ,Humans ,Receptors, Immunologic ,Adaptor Proteins, Signal Transducing ,Immune Evasion ,030304 developmental biology ,0303 health sciences ,NS3 ,Innate immune system ,biology ,virus diseases ,MDA5 ,biochemical phenomena, metabolism, and nutrition ,Hepatitis C ,Immunity, Innate ,digestive system diseases ,3. Good health ,Ubiquitin ligase ,HEK293 Cells ,Viral replication ,Insect Science ,Hepatocytes ,biology.protein ,DEAD Box Protein 58 ,Pathogenesis and Immunity ,Interferon Regulatory Factor-3 ,030211 gastroenterology & hepatology ,Serine Proteases ,Signal transduction ,IRF3 ,Signal Transduction ,medicine.drug - Abstract
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 the addition of Riplet to these cells reduced HCV Y16F replication, whereas the addition of Riplet lacking the RING domain restored HCV Y16F replication. In addition, TBK1 inhibition or 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 noncanonical Riplet-TBK1-IRF3-dependent, but RIG-I-MAVS-independent, signaling pathway that limits HCV infection. IMPORTANCE The 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. Since 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.
- Published
- 2019
39. RAB1B interacts with TRAF3 to promote antiviral innate immunity
- Author
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Moonhee Park, Graham D. Williams, Dillon J. Fernando, Madhuvanthi Vijayan, Stacy M. Horner, and Dia C. Beachboard
- Subjects
TRAF3 ,0303 health sciences ,Innate immune system ,Pattern recognition receptor ,Signal transducing adaptor protein ,GTPase ,Biology ,3. Good health ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,TANK-binding kinase 1 ,Sense (molecular biology) ,IRF3 ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Nucleic acid-based antiviral innate immunity activates a signaling cascade that induces type I and type III interferons (IFNs), and other cytokines. This signaling, which is highly regulated, is initiated by pattern recognition receptors, such as RIG-I, that sense viral RNA and then signal to the adaptor protein, MAVS. This adaptor protein then recruits additional signaling proteins, including TRAF3 and TBK1, to form a signaling complex that results in IRF3 activation for transcriptional induction of IFN. Here, we show that the GTPase trafficking protein RAB1B positively regulates RIG-I signaling to promote IFN-β induction and the antiviral response. Over-expression of RAB1B increases RIG-I-mediated signaling to IFN-β, while deletion results in reduced signaling of this pathway. Additionally, this loss of RAB1B results in a dampened antiviral response, as Zika virus infection is enhanced in the absence of RAB1B. Importantly, we identified the mechanism of RAB1B action by determining that it interacts with TRAF3 to facilitate the interaction of TRAF3 with MAVS. Thus, we identified RAB1B as a regulator of TRAF3 to promote the formation of innate immune signaling complexes in response to nucleic acid sensing.
- Published
- 2019
40. Measuring Hepatitis C Virus Envelopment by Using a Proteinase K Protection Assay
- Author
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Allison E. Roder and Stacy M. Horner
- Subjects
Hepatitis C virus ,viruses ,Immunoblotting ,Cell Culture Techniques ,Host factors ,Hepacivirus ,medicine.disease_cause ,Article ,Cell Line ,03 medical and health sciences ,medicine ,Humans ,Envelopment ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,Chemistry ,Electroporation ,Viral Core Proteins ,Virus Assembly ,030302 biochemistry & molecular biology ,Viral nucleocapsid ,Virion ,Proteinase K ,Virology ,Hepatitis C ,Virion assembly ,biology.protein ,RNA, Viral ,Endopeptidase K ,Glycoprotein - Abstract
The infectious virion of hepatitis C virus (HCV) is made up of the viral nucleocapsid surrounded by an envelope that contains an ER-derived membrane bilayer, cellular lipids, and the viral E1 and E2 glycoproteins. Because the infectious HCV particle contains both protein and lipid layers, selective disruption of these layers and analysis for the presence or absence of resulting virion components can be used to study the virion assembly process. This chapter describes an experimental method to measure HCV virion envelopment, which can reveal the mechanisms of how specific viral protein-protein interactions and host factors contribute to the process of HCV envelopment.
- Published
- 2018
41. Innate immune evasion strategies of DNA and RNA viruses
- Author
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Dia C. Beachboard and Stacy M. Horner
- Subjects
0301 basic medicine ,Microbiology (medical) ,viruses ,Biology ,Microbiology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Immune system ,Humans ,RNA Viruses ,Receptors, Immunologic ,Gene ,Transcription factor ,Immune Evasion ,Innate immune system ,Pattern recognition receptor ,DNA Viruses ,RNA ,Virology ,Immunity, Innate ,3. Good health ,030104 developmental biology ,Infectious Diseases ,chemistry ,Receptors, Pattern Recognition ,Host-Pathogen Interactions ,Interferon Type I ,DEAD Box Protein 58 ,Signal transduction ,DNA ,Signal Transduction - Abstract
Upon infection, both DNA and RNA viruses can be sensed by pattern recognition receptors (PRRs) in the cytoplasm or the nucleus to activate antiviral innate immunity. Sensing of viral products leads to the activation of a signaling cascade that ultimately results in transcriptional activation of type I and III interferons, as well as other antiviral genes that together mediate viral clearance and inhibit viral spread. Therefore, in order for viruses to replicate and spread efficiently, they must inhibit the host signaling pathways that induce the innate antiviral immune response. In this review, we will highlight recent advances in the understanding of the mechanisms by which viruses evade PRR detection, intermediate signaling molecule activation, transcription factor activation, and the actions of antiviral proteins.
- Published
- 2016
- Full Text
- View/download PDF
42. Cooperation between the Hepatitis C Virus p7 and NS5B Proteins Enhances Virion Infectivity
- Author
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Allison E. Roder, Stacy M. Horner, and Mounavya Aligeti
- Subjects
viruses ,Hepatitis C virus ,Immunology ,RNA-dependent RNA polymerase ,Hepacivirus ,Viral Nonstructural Proteins ,Biology ,Virus Replication ,medicine.disease_cause ,Microbiology ,Virus ,Viral Proteins ,chemistry.chemical_compound ,Cell Line, Tumor ,Virology ,medicine ,Humans ,NS5B ,Infectivity ,Base Sequence ,Sequence Analysis, RNA ,Virus Assembly ,Structure and Assembly ,Virion ,RNA ,Lipid Droplets ,Viral Load ,RNA-Dependent RNA Polymerase ,Hepatitis C ,Sphingomyelins ,chemistry ,Viral replication ,Insect Science ,RNA, Viral ,Lipoproteins, HDL ,Viral load - Abstract
The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly. IMPORTANCE Hepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection.
- Published
- 2015
43. Altered m6A Modification of Specific Cellular Transcripts Affects Flaviviridae Infection
- Author
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Helen M. Lazear, Alexa B. R. McIntyre, Christopher E. Mason, Nandan S. Gokhale, Melissa D. Mattocks, Christopher L. Holley, and Stacy M. Horner
- Subjects
0303 health sciences ,Innate immune system ,viruses ,Hepatitis C virus ,Alternative splicing ,Translation (biology) ,Cell Biology ,Dengue virus ,Biology ,medicine.disease_cause ,biology.organism_classification ,Virology ,CIRBP ,3. Good health ,Transcriptome ,03 medical and health sciences ,Flaviviridae ,0302 clinical medicine ,medicine ,Unfolded protein response ,Molecular Biology ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
SummaryThe 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.
- Published
- 2020
44. Pervasive tertiary structure in the dengue virus RNA genome
- Author
-
Nandan S. Gokhale, Michael J. McFadden, Mark A. Boerneke, Jules B. Weinstein, Brejnev M. Muhire, Darren P. Martin, Kevin M. Weeks, William B. Messer, Matthew T. Sacco, Stacy M. Horner, and Elizabeth A. Dethoff
- Subjects
0301 basic medicine ,Models, Molecular ,Computational biology ,Genome, Viral ,Dengue virus ,medicine.disease_cause ,Serogroup ,Genome ,03 medical and health sciences ,Capsid ,medicine ,Nucleotide ,Base Pairing ,chemistry.chemical_classification ,Multidisciplinary ,030102 biochemistry & molecular biology ,biology ,Nucleic acid tertiary structure ,Virus Assembly ,RNA ,RNA virus ,Biological Sciences ,Dengue Virus ,Non-coding RNA ,biology.organism_classification ,Protein tertiary structure ,030104 developmental biology ,chemistry ,Nucleic Acid Conformation ,RNA, Viral ,Genetic Fitness - Abstract
RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds—in which helices pack together to form complex 3D structures—and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.
- Published
- 2018
45. An atlas of genetic variation linking pathogen-induced cellular traits to human disease
- Author
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Stacy M. Horner, Jeffrey R. Barker, Soo Chan Lee, Robert J. Carroll, Mark R. DeLong, Joseph Heitman, Liuyang Wang, Kelly J. Pittman, Joshua C. Denny, Kyle D. Gibbs, Graham D. Williams, Tom Balmat, Anusha M. Gopalakrishnan, Andy Ingham, Alejandro L. Antonia, Dennis C. Ko, Raphael H. Valdivia, Ian B. Stanaway, David R. Crosslin, Gail P. Jarvik, and Raul E. Salinas
- Subjects
0301 basic medicine ,DNA Replication ,Hepatitis, Viral, Human ,DNA Mutational Analysis ,Genome-wide association study ,Nerve Tissue Proteins ,Biology ,Phenome ,Web Browser ,Infections ,Microbiology ,Article ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,Risk Factors ,Virology ,Genetic variation ,Databases, Genetic ,SNP ,Electronic Health Records ,Humans ,Genetic Predisposition to Disease ,Gene ,Genetic association ,Genetics ,Genome, Human ,Data Collection ,Antibodies, Monoclonal ,Computational Biology ,Genetic Variation ,Genetic Pleiotropy ,Inflammatory Bowel Diseases ,Phenotype ,Chemokine CXCL10 ,030104 developmental biology ,Cytokines ,Parasitology ,Human genome ,030217 neurology & neurosurgery ,Genome-Wide Association Study ,Transcription Factors - Abstract
Pathogens 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) comprised of 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.
- Published
- 2018
46. The acidic domain of the hepatitis C virus NS4A protein is required for viral assembly and envelopment through interactions with the viral E1 glycoprotein
- Author
-
Allison E. Roder and Stacy M. Horner
- Subjects
chemistry.chemical_classification ,Mutation ,Chemistry ,Hepatitis C virus ,viruses ,Mutagenesis (molecular biology technique) ,RNA ,Transfection ,medicine.disease_cause ,Virology ,Protein–protein interaction ,Viral envelope ,medicine ,Glycoprotein - Abstract
Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and 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.Author SummaryRNA viruses, which encompass both established and emerging pathogens, pose significant public health challenges. Viruses in the familyFlavivirdae, including Dengue virus, Zika virus and hepatitis C virus (HCV), continue to cause morbidity and mortality worldwide. One HCV protein, NS4A, has known functions in several steps of the viral lifecycle, however, how it contributes to viral particle production is not understood. Here, we investigated the role of one region of NS4A, the C-terminal acidic domain, in regulating the viral lifecycle. We found that some of the amino acids within this domain are important for viral envelopment to make infectious particles, specifically through interaction with the E1 glycoprotein. NS4A interacts with the first hydrophobic domain of E1. Disruption of this interaction prevents the production of infectious virus particles and instead results in release of low density Core protein complexes that lack HCV RNA into the cellular supernatant. Overall, our results reveal that NS4A is important for late stages of the HCV lifecycle and suggest that the interaction between NS4A and E1 may regulate the incorporation of viral RNA into the virion for the formation of infectious HCV particles.
- Published
- 2018
- Full Text
- View/download PDF
47. A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time
- Author
-
Allison E. Roder, Stacy M. Horner, Debra L. Silver, Christine Vazquez, Aaron Mitchell-Dick, John J. McMahon, Kevin F. Labagnara, and Michael J. McFadden
- Subjects
0301 basic medicine ,Microcephaly ,Zika virus (ZIKV) ,NS2B-NS3 ,NS4B-NS5 ,reporter ,fluorescence ,live cell imaging ,apoptosis ,Cytological Techniques ,Green Fluorescent Proteins ,Active Transport, Cell Nucleus ,lcsh:QR1-502 ,Biology ,Viral Nonstructural Proteins ,Article ,lcsh:Microbiology ,Zika virus ,Cell Line ,Pathogenesis ,03 medical and health sciences ,Fluorescent cell ,Live cell imaging ,Genes, Reporter ,Virology ,medicine ,Animals ,Humans ,Cell Nucleus ,Fluorescent reporter ,Cell Death ,Zika Virus Infection ,Optical Imaging ,Serine Endopeptidases ,Zika Virus ,medicine.disease ,biology.organism_classification ,3. Good health ,Flavivirus ,030104 developmental biology ,Infectious Diseases ,Microscopy, Fluorescence ,Sexual contact ,Plasmids - Abstract
Zika virus (ZIKV) is a re-emerging flavivirus that is transmitted to humans through the bite of an infected mosquito or through sexual contact with an infected partner. ZIKV infection during pregnancy has been associated with numerous fetal abnormalities, including prenatal lethality and microcephaly. However, until recent outbreaks in the Americas, ZIKV has been relatively understudied, and therefore the biology and pathogenesis of ZIKV infection remain incompletely understood. Better methods to study ZIKV infection in live cells could enhance our understanding of the biology of ZIKV and the mechanisms by which ZIKV contributes to fetal abnormalities. To this end, we developed a fluorescent cell-based reporter system allowing for live imaging of ZIKV-infected cells. This system utilizes the protease activity of the ZIKV non-structural proteins 2B and 3 (NS2B-NS3) to specifically mark virus-infected cells. Here, we demonstrate the utility of this fluorescent reporter for identifying cells infected by ZIKV strains of two lineages. Further, we use this system to determine that apoptosis is induced in cells directly infected with ZIKV in a cell-autonomous manner. Ultimately, approaches that can directly track ZIKV-infected cells at the single cell-level have the potential to yield new insights into the host-pathogen interactions that regulate ZIKV infection and pathogenesis.
- Published
- 2018
48. Posttranscriptional m6A Editing of HIV-1 mRNAs Enhances Viral Gene Expression
- Author
-
Hal P. Bogerd, Bryan R. Cullen, Brigid Chiyoko Poling, Edward M. Kennedy, Nandan S. Gokhale, Delta Ghoshal, Anand V.R. Kornepati, Dong Kang, Stacy M. Horner, Kevin Tsai, and Joy B. Marshall
- Subjects
0301 basic medicine ,Untranslated region ,CD4-Positive T-Lymphocytes ,Gene Expression Regulation, Viral ,Human Immunodeficiency Virus Proteins ,RNA-binding protein ,Genome, Viral ,Biology ,Virus Replication ,Microbiology ,Article ,Cell Line ,03 medical and health sciences ,Virology ,Humans ,RNA, Messenger ,Cloning, Molecular ,3' Untranslated Regions ,Regulation of gene expression ,030102 biochemistry & molecular biology ,Three prime untranslated region ,HEK 293 cells ,RNA ,RNA-Binding Proteins ,Molecular biology ,030104 developmental biology ,HEK293 Cells ,Viral replication ,RNA editing ,HIV-1 ,RNA, Viral ,Parasitology ,RNA Editing - Abstract
Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression.
- Published
- 2017
49. Insights into antiviral innate immunity revealed by studying hepatitis C virus
- Author
-
Stacy M. Horner
- Subjects
Hepatitis C virus ,Immunology ,Context (language use) ,Hepacivirus ,medicine.disease_cause ,Biochemistry ,Article ,Virus ,Antiviral immunity ,Immune system ,Interferon ,medicine ,Animals ,Humans ,Immunology and Allergy ,Molecular Biology ,Immune Evasion ,Innate immune system ,biology ,RNA virus ,Hematology ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Hepatitis C ,Virology ,Immunity, Innate ,Interferon Type I ,bacteria ,medicine.drug - Abstract
Experimental studies on the interactions of the positive strand RNA virus hepatitis C virus (HCV) with the host have contributed to several discoveries in the field of antiviral innate immunity. These include revealing the antiviral sensing pathways that lead to the induction of type I interferon (IFN) during HCV infection and also the importance of type III IFNs in the antiviral immune response to HCV. These studies on HCV/host interactions have contributed to our overall understanding of viral sensing and viral evasion of the antiviral intracellular innate immune response. In this review, I will highlight how these studies of HCV/host interactions have led to new insights into antiviral innate immunity. Overall, I hope to emphasize that studying antiviral immunity in the context of virus infection is necessary to fully understand antiviral immunity and how it controls the outcome of viral infection.
- Published
- 2015
50. MAVS Coordination of Antiviral Innate Immunity
- Author
-
Christine Vazquez and Stacy M. Horner
- Subjects
Gem ,Innate immune system ,biology ,Endoplasmic reticulum ,Immunology ,Signal transducing adaptor protein ,RNA virus ,Endoplasmic Reticulum ,Subcellular localization ,biology.organism_classification ,Models, Biological ,Microbiology ,Virology ,Immunity, Innate ,Mitochondria ,Cell biology ,Immunity ,Cytoplasm ,Insect Science ,Host-Pathogen Interactions ,RNA Viruses ,Signal transduction ,Adaptor Proteins, Signal Transducing ,Signal Transduction - Abstract
RNA virus infection is sensed in the cytoplasm by the retinoic acid-inducible gene I (RIG-I)-like receptors. These proteins signal through the host adaptor protein MAVS to trigger the antiviral innate immune response. Here, we describe how MAVS subcellular localization impacts its function and the regulation underlying MAVS signaling. We propose a model to describe how the coordination of MAVS functions at the interface between the mitochondria and the mitochondrion-associated endoplasmic reticulum (ER) membrane programs antiviral signaling.
- Published
- 2015
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