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1.
J Mol Biol ; : 167247, 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34537236

RESUMO

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 fat mass and obesity-associated protein (FTO) in the type I IFN response. FTO, which can remove either m6A or 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 requires its catalytic activity but is not through the actions of FTO on m6Am. Interestingly, depletion of FTO led to activation of the transcription factor STAT3, 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.

2.
Immunol Rev ; 304(1): 169-180, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34405413

RESUMO

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.

3.
PLoS Biol ; 19(7): e3001342, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329302

RESUMO

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.


Assuntos
Adenosina Desaminase , RNA de Cadeia Dupla , Adenosina , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Interferons/metabolismo , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
4.
Sci Immunol ; 6(61)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215679

RESUMO

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/EBPß and C/EBPδ in response to IL-17A, as well as IL-17F and TNFα. Prompted by the observation that C/EBPß/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/EBPß/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/EBPß/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.

5.
Cell Rep ; 34(9): 108798, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33657363

RESUMO

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 controls 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.

6.
Brief Funct Genomics ; 20(2): 86-93, 2021 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-33401298

RESUMO

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.


Assuntos
RNA Viral , Proteínas de Ligação a RNA , Adenosina , RNA Mensageiro , RNA Viral/genética
7.
Trends Biochem Sci ; 46(5): 366-377, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33309325

RESUMO

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.


Assuntos
Viroses , Adenosina/análogos & derivados , Citocinas , Humanos , Imunidade Inata , RNA Viral
8.
Cell ; 183(5): 1185-1201.e20, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33242417

RESUMO

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.


Assuntos
Genômica , Mitocôndrias/patologia , Voo Espacial , Estresse Fisiológico , Animais , Ritmo Circadiano , Matriz Extracelular/metabolismo , Humanos , Imunidade Inata , Metabolismo dos Lipídeos , Análise do Fluxo Metabólico , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Músculos/imunologia , Especificidade de Órgãos , Olfato/fisiologia
9.
Nat Commun ; 11(1): 6016, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-33243990

RESUMO

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.


Assuntos
Adenosina/análogos & derivados , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Splicing de RNA , RNA Viral/metabolismo , Células A549 , Adenosina/metabolismo , Adenovírus Humanos/patogenicidade , DNA Viral/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , Análise de Sequência de RNA , Replicação Viral
10.
bioRxiv ; 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32511352

RESUMO

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused thousands of deaths worldwide, including >18,000 in New York City (NYC) alone. The sudden emergence of this pandemic has highlighted a pressing clinical need for rapid, scalable diagnostics that can detect infection, interrogate strain evolution, and identify novel patient biomarkers. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs, plus a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, bacterial, and viral profiling. We applied both technologies across 857 SARS-CoV-2 clinical specimens and 86 NYC subway samples, providing a broad molecular portrait of the COVID-19 NYC outbreak. Our results define new features of SARS-CoV-2 evolution, nominate a novel, NYC-enriched viral subclade, reveal specific host responses in interferon, ACE, hematological, and olfaction pathways, and examine risks associated with use of ACE inhibitors and angiotensin receptor blockers. Together, these findings have immediate applications to SARS-CoV-2 diagnostics, public health, and new therapeutic targets.

11.
mSphere ; 5(3)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404510

RESUMO

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.


Assuntos
Regulação da Expressão Gênica/imunologia , Interferon Tipo I/imunologia , Metiltransferases/genética , RNA Mensageiro/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular , Células HEK293 , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metilação , Biossíntese de Proteínas , Vírus de RNA/imunologia , Vírus de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Células THP-1 , Replicação Viral/imunologia
12.
Sci Rep ; 10(1): 6590, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313079

RESUMO

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.


Assuntos
Adenosina/genética , RNA Mensageiro/isolamento & purificação , RNA/isolamento & purificação , Algoritmos , Sequência de Bases , Humanos , Imunoprecipitação , Metilação , RNA/genética , RNA Mensageiro/genética , Análise de Sequência de RNA , Software
13.
Sci Adv ; 6(14): eaay3245, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32270034

RESUMO

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.


Assuntos
Resistência à Doença , Interações Hospedeiro-Patógeno , Imunidade Inata , Interleucinas/metabolismo , Transdução de Sinais , Infecção por Zika virus/etiologia , Infecção por Zika virus/metabolismo , Zika virus/imunologia , Biomarcadores , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Resistência à Doença/imunologia , Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/virologia , Fator de Transcrição STAT1/metabolismo , Pele/imunologia , Pele/metabolismo , Pele/virologia
14.
Mol Cell ; 77(3): 542-555.e8, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31810760

RESUMO

The RNA modification N6-methyladenosine (m6A) modulates mRNA fate and thus affects many biological processes. We analyzed m6A 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, viral activation of innate immune sensing or the endoplasmic reticulum (ER) stress response contributes to the changes in m6A in RIOK3 or 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.


Assuntos
Adenosina/análogos & derivados , Infecções por Flaviviridae/genética , RNA Mensageiro/genética , Adenosina/genética , Linhagem Celular , Dengue/virologia , Vírus da Dengue/genética , Flaviviridae/genética , Hepacivirus/genética , Hepatite C/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/genética , Zika virus/genética , Infecção por Zika virus/genética
15.
J Virol ; 93(23)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31534039

RESUMO

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.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C/virologia , Serina Proteases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Proteína DEAD-box 58/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Hepatócitos/virologia , Humanos , Evasão da Resposta Imune , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Proteínas Serina-Treonina Quinases , Replicação Viral
16.
J Biol Chem ; 294(39): 14231-14240, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31375559

RESUMO

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-ß induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-ß 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.


Assuntos
Imunidade Inata , Fator 3 Associado a Receptor de TNF/metabolismo , Infecção por Zika virus/imunologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Interferon beta/metabolismo , Ligação Proteica , Transdução de Sinais , Células Vero
17.
Annu Rev Virol ; 6(1): 235-253, 2019 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-31283446

RESUMO

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.


Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica , Interações entre Hospedeiro e Microrganismos , RNA Viral/química , Imunidade Adaptativa , Adenosina/química , Animais , Humanos , Imunidade Inata , Camundongos , Processamento Pós-Transcricional do RNA , RNA Viral/genética , Viroses/imunologia , Replicação Viral
18.
PLoS Pathog ; 15(2): e1007163, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30730994

RESUMO

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.


Assuntos
Proteínas de Transporte/metabolismo , Hepacivirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C Crônica/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Domínios Proteicos , RNA Viral , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Vírion/metabolismo , Vírion/fisiologia , Montagem de Vírus , Replicação Viral
19.
Methods Mol Biol ; 1911: 209-217, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593628

RESUMO

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.


Assuntos
Endopeptidase K/metabolismo , Hepacivirus/fisiologia , Hepatite C/virologia , Proteínas do Core Viral/metabolismo , Vírion/fisiologia , Técnicas de Cultura de Células/métodos , Linhagem Celular , Eletroporação/métodos , Hepacivirus/genética , Humanos , Immunoblotting/métodos , RNA Viral/genética , Proteínas do Core Viral/genética , Vírion/genética , Montagem de Vírus
20.
Proc Natl Acad Sci U S A ; 115(45): 11513-11518, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30341219

RESUMO

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.


Assuntos
Capsídeo/ultraestrutura , Vírus da Dengue/ultraestrutura , Genoma Viral , RNA Viral/ultraestrutura , Pareamento de Bases , Capsídeo/química , Capsídeo/metabolismo , Vírus da Dengue/classificação , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Aptidão Genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Sorogrupo , Montagem de Vírus/genética
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