RESUMO
Some negative-sense RNA viruses, including measles virus (MeV), share the characteristic that during their infection cycle, cytoplasmic inclusion bodies (IBs) are formed where components of the viral replication machinery are concentrated. As a foci of viral replication, how IBs act to enhance the efficiency of infection by affecting virus-host interactions remains an important topic of investigation. We previously established that upon MeV infection, the epigenetic host protein, WD repeat-containing protein 5 (WDR5), translocates to cytoplasmic viral IBs and facilitates MeV replication. We now show that WDR5 is recruited to IBs by forming a complex with IB-associated MeV phosphoprotein via a conserved binding motif located on the surface of WDR5. Furthermore, we provide evidence that WDR5 promotes viral replication by suppressing a major innate immune response pathway, the double-stranded RNA-mediated activation of protein kinase R and integrated stress response. IMPORTANCE: MeV is a pathogen that remains a global concern, with an estimated 9 million measles cases and 128,000 measles deaths in 2022 according to the World Health Organization. A large population of the world still has inadequate access to the effective vaccine against the exceptionally transmissible MeV. Measles disease is characterized by a high morbidity in children and in immunocompromised individuals. An important area of research for negative-sense RNA viruses, including MeV, is the characterization of the complex interactome between virus and host occurring at cytoplasmic IBs where viral replication occurs. Despite the progress made in understanding IB structures, little is known regarding the virus-host interactions within IBs and the role of these interactions in promoting viral replication and antagonizing host innate immunity. Herein we provide evidence suggesting a model by which MeV IBs utilize the host protein WDR5 to suppress the protein kinase R-integrated stress response pathway.
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A novel coronavirus now known as SARS-CoV-2 emerged in late 2019, possibly following a zoonotic crossover from a coronavirus present in bats. This virus was identified as the pathogen responsible for the severe respiratory disease, coronavirus disease-19 (COVID-19), which as of May 2023, has killed an estimated 6.9 million people globally according to the World Health Organization. The interferon (IFN) response, a cornerstone of antiviral innate immunity, plays a key role in determining the outcome of infection by SARS-CoV-2. This review considers evidence that SARS-CoV-2 infection leads to IFN production; that virus replication is sensitive to IFN antiviral action; molecular mechanisms by which the SARS-CoV-2 virus antagonizes IFN action; and how genetic variability of SARS-CoV-2 and the human host affects the IFN response at the level of IFN production or action or both. Taken together, the current understanding suggests that deficiency of an effective IFN response is an important determinant underlying some cases of critical COVID-19 disease and that IFNλ and IFNα/ß have potential as therapeutics for the treatment of SARS-CoV-2 infection.
Assuntos
COVID-19 , Interferon Tipo I , Humanos , COVID-19/genética , SARS-CoV-2 , Linhagem Celular , Antivirais/farmacologia , Antivirais/uso terapêutico , Interferon-alfa , Imunidade Inata , Interferon Tipo I/genéticaRESUMO
BACKGROUND: Antimicrobial resistance (AMR) poses a critical threat to public health and disproportionately affects the health and well-being of persons in low-income and middle-income countries. Our aim was to identify synthetic antimicrobials termed conjugated oligoelectrolytes (COEs) that effectively treated AMR infections and whose structures could be readily modified to address current and anticipated patient needs. METHODS: Fifteen chemical variants were synthesized that contain specific alterations to the COE modular structure, and each variant was evaluated for broad-spectrum antibacterial activity and for in vitro cytotoxicity in cultured mammalian cells. Antibiotic efficacy was analyzed in murine models of sepsis; in vivo toxicity was evaluated via a blinded study of mouse clinical signs as an outcome of drug treatment. FINDINGS: We identified a compound, COE2-2hexyl, that displayed broad-spectrum antibacterial activity. This compound cured mice infected with clinical bacterial isolates derived from patients with refractory bacteremia and did not evoke bacterial resistance. COE2-2hexyl has specific effects on multiple membrane-associated functions (e.g., septation, motility, ATP synthesis, respiration, membrane permeability to small molecules) that may act together to negate bacterial cell viability and the evolution of drug-resistance. Disruption of these bacterial properties may occur through alteration of critical protein-protein or protein-lipid membrane interfaces-a mechanism of action distinct from many membrane disrupting antimicrobials or detergents that destabilize membranes to induce bacterial cell lysis. INTERPRETATION: The ease of molecular design, synthesis and modular nature of COEs offer many advantages over conventional antimicrobials, making synthesis simple, scalable and affordable. These COE features enable the construction of a spectrum of compounds with the potential for development as a new versatile therapy for an imminent global health crisis. FUNDING: U.S. Army Research Office, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute.
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Anti-Infecciosos , Infecções Bacterianas , Sepse , Camundongos , Animais , Antibacterianos/farmacologia , Infecções Bacterianas/microbiologia , Anti-Infecciosos/farmacologia , Bactérias , Sepse/tratamento farmacológico , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana Múltipla , MamíferosRESUMO
Many negative-sense RNA viruses, including measles virus (MeV), are thought to carry out much of their viral replication in cytoplasmic membraneless foci known as inclusion bodies (IBs). The mechanisms by which IBs facilitate efficient viral replication remain largely unknown but may involve an intricate network of regulation at the host-virus interface. Viruses are able to modulate such interactions by a variety of strategies including adaptation of their genomes and "hijacking" of host proteins. The latter possibility broadens the molecular reservoir available for a virus to enhance its replication and/or antagonize host antiviral responses. Here, we show that the cellular 5'-3' exoribonuclease, XRN1, is a host protein hijacked by MeV. We found that upon MeV infection, XRN1 is translocated to cytoplasmic IBs where it acts in a proviral manner by preventing the accumulation of double-stranded RNA (dsRNA) within the IBs. This leads to the suppression of the dsRNA-induced innate immune responses mediated via the protein kinase R (PKR)-integrated stress response (ISR) pathway. IMPORTANCE Measles virus remains a major global health threat due to its high transmissibility and significant morbidity in children and immunocompromised individuals. Although there is an effective vaccine against MeV, a large population in the world remains without access to the vaccine, contributing to more than 7,000,000 measles cases and 60,000 measles deaths in 2020 (CDC). For negative-sense RNA viruses including MeV, one active research area is the exploration of virus-host interactions occurring at cytoplasmic IBs where viral replication takes place. In this study we present evidence suggesting a model in which MeV IBs antagonize host innate immunity by recruiting XRN1 to reduce dsRNA accumulation and subsequent PKR kinase activation/ISR induction. In the absence of XRN1, the increased dsRNA level acts as a potent activator of the antiviral PKR/ISR pathway leading to suppression of global cap-dependent mRNA translation and inhibition of viral replication.
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Exorribonucleases , Sarampo , Proteínas Associadas aos Microtúbulos , Replicação Viral , Humanos , eIF-2 Quinase/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Sarampo/genética , Sarampo/virologia , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Provírus/genética , RNA de Cadeia Dupla , Corpos de Inclusão ViralRESUMO
Importance: A critical need exists in low-income and middle-income countries for low-cost, low-tech, yet highly reliable and scalable testing for SARS-CoV-2 virus that is robust against circulating variants. Objective: To assess whether a smartphone-based assay is suitable for SARS-CoV-2 and influenza virus testing without requiring specialized equipment, accessory devices, or custom reagents. Design, Setting, and Participants: This cohort study enrolled 2 subgroups of participants (symptomatic and asymptomatic) at Santa Barbara Cottage Hospital. The symptomatic group consisted of 20 recruited patients who tested positive for SARS-CoV-2 with symptoms; 30 asymptomatic patients were recruited from the same community, through negative admission screening tests for SARS-CoV-2. The smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) was first optimized for analysis of human saliva samples spiked with either SARS-CoV-2 or influenza A or B virus; these results then were compared with those obtained by side-by-side analysis of spiked samples using the Centers for Disease Control and Prevention (CDC) criterion-standard reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay. Next, both assays were used to test for SARS-CoV-2 and influenza viruses present in blinded clinical saliva samples obtained from 50 hospitalized patients. Statistical analysis was performed from May to June 2021. Exposures: Testing for SARS-CoV-2 and influenza A and B viruses. Main Outcomes and Measures: SARS-CoV-2 and influenza infection status and quantitative viral load were determined. Results: Among the 50 eligible participants with no prior SARS-CoV-2 infection included in the study, 29 were men. The mean age was 57 years (range, 21 to 93 years). SmaRT-LAMP exhibited 100% concordance (50 of 50 patient samples) with the CDC criterion-standard diagnostic for SARS-CoV-2 sensitivity (20 of 20 positive and 30 of 30 negative) and for quantitative detection of viral load. This platform also met the CDC criterion standard for detection of clinically similar influenza A and B viruses in spiked saliva samples (n = 20), and in saliva samples from hospitalized patients (50 of 50 negative). The smartphone-based LAMP assay was rapid (25 minutes), sensitive (1000 copies/mL), low-cost (<$7/test), and scalable (96 samples/phone). Conclusions and Relevance: In this cohort study of saliva samples from patients, the smartphone-based LAMP assay detected SARS-CoV-2 infection and exhibited concordance with RT-qPCR tests. These findings suggest that this tool could be adapted in response to novel CoV-2 variants and other pathogens with pandemic potential including influenza and may be useful in settings with limited resources.
Assuntos
COVID-19 , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Orthomyxoviridae/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Smartphone , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estados Unidos , Adulto JovemRESUMO
C6 deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA) is catalyzed by a family of enzymes known as ADARs (adenosine deaminases acting on RNA) encoded by three genes in mammals. Alternative promoters and splicing produce two ADAR1 proteins, an interferon-inducible cytoplasmic p150 and a constitutively expressed p110 that like ADAR2 is a nuclear enzyme. ADAR3 lacks deaminase activity. A-to-I editing occurs with both viral and cellular RNAs. Deamination activity is dependent on dsRNA substrate structure and regulatory RNA-binding proteins and ranges from highly site selective with hepatitis D RNA and glutamate receptor precursor messenger RNA (pre-mRNA) to hyperediting of measles virus and polyomavirus transcripts and cellular inverted Alu elements. Because I base-pairs as guanosine instead of A, editing can alter mRNA decoding, pre-mRNA splicing, and microRNA silencing. Editing also alters dsRNA structure, thereby suppressing innate immune responses including interferon production and action.
Assuntos
Adenosina Desaminase , Viroses , Adenosina Desaminase/química , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Edição de RNA , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Liquid-liquid phase separation (LLPS) represents a major physiochemical principle to organize intracellular membrane-less structures. Studies with non-segmented negative-sense (NNS) RNA viruses have uncovered a key role of LLPS in the formation of viral inclusion bodies (IBs), sites of viral protein concentration in the cytoplasm of infected cells. These studies further reveal the structural and functional complexity of viral IB factories and provide a foundation for their future research. Herein, we review the literature leading to the discovery of LLPS-driven formation of IBs in NNS RNA virus-infected cells and the identification of viral scaffold components involved, and then outline important questions and challenges for IB assembly and disassembly. We discuss the functional implications of LLPS in the life cycle of NNS RNA viruses and host responses to infection. Finally, we speculate on the potential mechanisms underlying IB maturation, a phenomenon relevant to many human diseases.
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Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , RNA Viral , Animais , Interações Hospedeiro-Patógeno , Humanos , Extração Líquido-Líquido , Vírus de RNA/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fenômenos Fisiológicos Virais , Replicação ViralRESUMO
The role of RNA-dependent protein kinase R (PKR) and its association with misfolded protein expression in cancer cells are unclear. Herein we report that PKR regulates misfolded protein clearance by preventing it release through exosomes and promoting lysosomal degradation of misfolded prion proteins in cancer cells. We demonstrated that PKR contributes to the lysosome function and regulates misfolded prion protein clearance. We hypothesized that PKR-associated lysosome function is critical for cancer but not normal cell survival, representing an effective approach for highly targeted cancer therapy. In screening a compound library, we identified two PKR-associated compounds 1 and 2 (Pac 1 and 2) did not affect normal cells but selectively induced cell death in cancer cells depending on their PKR expression status. Pac 1 significantly inhibited the growth of human lung and breast xenograft tumors in mice with no toxicity. Pac 1 binds to PI4K2A and disrupts the PKR/PI4K2A-associated lysosome complex, contributing to destabilization of cancer cell lysosomes and triggering cell death. We observed that PKR and PI4K2A play significant prognostic roles in breast cancer patients. These results demonstrate that targeting of a PI4K2A/PKR lysosome complex may be an effective approach for cancer therapy.
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Antineoplásicos/farmacologia , Lisossomos/metabolismo , Neoplasias/tratamento farmacológico , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Proteólise , Resposta a Proteínas não Dobradas , eIF-2 Quinase/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular , Bases de Dados Genéticas/estatística & dados numéricos , Exossomos/metabolismo , Feminino , Humanos , Camundongos , Camundongos SCID , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prognóstico , Dobramento de Proteína , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/metabolismoRESUMO
We report on the discovery of a new organized lipid-nucleic acid phase upon intercalation of blunt duplexes of short DNA (sDNA) within cationic multilayer fluid membranes. End-to-end interactions between sDNA leads to columnar stacks. At high membrane charge density, with the inter-sDNA column spacing (dsDNA) comparable but larger than the diameter of sDNA, a 2D columnar phase (i.e., a 2D smectic) is found similar to the phase in cationic liposome-DNA complexes with long lambda-phage DNA. Remarkably, with increasing dsDNA as the membrane charge density is lowered, a transition is observed to a 3D columnar phase of stacked sDNA. This occurs even though direct DNA-DNA electrostatic interactions across layers are screened by diffusing cationic lipids near the phosphate groups of sDNA. Softening of the membrane bending rigidity (κ), which further promotes membrane undulations, significantly enhances the 3D columnar phase. These observations are consistent with a model by Schiessel and Aranda-Espinoza where local membrane undulations, due to electrostatically induced membrane wrapping around sDNA columns, phase lock from layer-to-layer, thereby precipitating coherent "crystal-like" undulations coupled to sDNA columns with long-range position and orientation order. The finding that this new phase is stable at large dsDNA and enhanced with decreasing κ is further supportive of the model where the elastic cost of membrane deformation per unit area around sDNA columns (â κh2/dsDNA4, h2 = sum of square of amplitudes of the inner and outer monolayer undulations) is strongly reduced relative to the favorable electrostatic attractions of partially wrapped membrane around sDNA columns. The findings have broad implications in the design of membrane-mediated assembly of functional nanoparticles in 3D.
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DNA/química , Ácidos Graxos Monoinsaturados/química , Fosfatidilcolinas/química , Compostos de Amônio Quaternário/química , Lipossomos/química , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Nonsegmented negative-strand RNA viruses, including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane bound, and they serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here, we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying that they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggests that Mononegavirales have broadly evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells.IMPORTANCE Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur, and globally â¼100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WD repeat-containing protein 5 (WDR5) enhances MeV growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here, we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies, that these structures display properties characteristic of phase-separated liquid organelles, and that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.
Assuntos
Corpos de Inclusão Viral/fisiologia , Vírus do Sarampo/fisiologia , Sarampo/virologia , Nucleoproteínas/metabolismo , Organelas/fisiologia , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Citoplasma/virologia , Células HeLa , Humanos , Corpos de Inclusão Viral/virologia , Extração Líquido-Líquido , Proteínas do Nucleocapsídeo , Nucleoproteínas/genética , Organelas/virologia , Fosfoproteínas/genética , Proteínas Virais/genéticaRESUMO
Herbert "Herb" Tabor, who celebrated his 100th birthday this past year, served the Journal of Biological Chemistry as a member of the Editorial Board beginning in 1961, as an Associate Editor, and as Editor-in-Chief for 40 years, from 1971 until 2010. Among the many discoveries in biological chemistry during this period was the identification of RNA modification by C6 deamination of adenosine (A) to produce inosine (I) in double-stranded (ds) RNA. This posttranscriptional RNA modification by adenosine deamination, known as A-to-I RNA editing, diversifies the transcriptome and modulates the innate immune interferon response. A-to-I editing is catalyzed by a family of enzymes, adenosine deaminases acting on dsRNA (ADARs). The roles of A-to-I editing are varied and include effects on mRNA translation, pre-mRNA splicing, and micro-RNA silencing. Suppression of dsRNA-triggered induction and action of interferon, the cornerstone of innate immunity, has emerged as a key function of ADAR1 editing of self (cellular) and nonself (viral) dsRNAs. A-to-I modification of RNA is essential for the normal regulation of cellular processes. Dysregulation of A-to-I editing by ADAR1 can have profound consequences, ranging from effects on cell growth and development to autoimmune disorders.
Assuntos
Adenosina Desaminase/metabolismo , Imunidade Inata/imunologia , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Humanos , Imunidade Inata/fisiologiaRESUMO
Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5-deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication.IMPORTANCE Measles virus is a human pathogen that remains a global concern, with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here, we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus.
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Citoplasma/virologia , Histona-Lisina N-Metiltransferase/metabolismo , Corpos de Inclusão Viral/fisiologia , Vírus do Sarampo/fisiologia , Sarampo/virologia , Proteínas Virais/metabolismo , Replicação Viral , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sarampo/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genéticaRESUMO
Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.
Assuntos
Adenosina Desaminase , Primatas/genética , Edição de RNA/genética , Proteínas de Ligação a RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Feminino , Genótipo , Células HEK293 , Humanos , Masculino , Camundongos , Músculos/metabolismo , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Proteólise , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise Espaço-Temporal , Especificidade da Espécie , Transcriptoma/genéticaRESUMO
UNLABELLED: Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as ß-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and ß-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and ß-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE: Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and ß-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.
Assuntos
Vírus da Febre do Vale do Rift/patogenicidade , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , RNA Interferente Pequeno , Vírus da Febre do Vale do Rift/química , Vírus da Febre do Vale do Rift/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Células Vero , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genética , Replicação Viral , Proteínas Contendo Repetições de beta-Transducina/deficiência , Proteínas Contendo Repetições de beta-Transducina/genética , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genéticaRESUMO
Adenosine deaminases acting on double-stranded RNA (ADARs) catalyze the deamination of adenosine (A) to produce inosine (I) in double-stranded (ds) RNA structures, a process known as A-to-I RNA editing. dsRNA is an important trigger of innate immune responses, including interferon (IFN) production and action. We examined the role of A-to-I RNA editing by two ADARs, ADAR1 and ADAR2, in the sensing of self-RNA in the absence of pathogen infection, leading to activation of IFN-induced, RNA-mediated responses in mouse embryo fibroblasts. IFN treatment of Adar1(-/-) cells lacking both the p110 constitutive and p150 IFN-inducible ADAR1 proteins induced formation of stress granules, whereas neither wild-type (WT) nor Adar2(-/-) cells displayed a comparable stress granule response following IFN treatment. Phosphorylation of protein synthesis initiation factor eIF2α at serine 51 was increased in IFN-treated Adar1(-/-) cells but not in either WT or Adar2(-/-) cells following IFN treatment. Analysis by deep sequencing of mouse exonic loci containing A-to-I-editing sites revealed that the majority of editing in mouse embryo fibroblasts was carried out by ADAR1. IFN treatment increased editing in both WT and Adar2(-/-) cells but not in either Adar1(-/-) or Adar1(-/-) (p150) cells or Stat1(-/-) or Stat2(-/-) cells. Hyper-edited sites found in predicted duplex structures showed strand bias of editing for some RNAs. These results implicate ADAR1 p150 as the major A-to-I editor in mouse embryo fibroblasts, acting as a feedback suppressor of innate immune responses otherwise triggered by self-RNAs possessing regions of double-stranded character.
Assuntos
Adenosina Desaminase/fisiologia , Imunidade Inata , Edição de RNA , RNA de Cadeia Dupla/metabolismo , Animais , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Desaminação , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos/metabolismo , Tolerância Imunológica , Interferon-alfa/fisiologia , Camundongos Knockout , Fosforilação , Processamento de Proteína Pós-Traducional , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/fisiologia , Transdução de SinaisRESUMO
Expression of adenosine deaminase acting on RNA1 (ADAR1) is driven by alternative promoters. Promoter PA, activated by interferon (IFN), produces transcripts that encode the inducible p150 ADAR1 protein, whereas PB specifies the constitutively expressed p110 protein. We show using Stat1(-/-), Stat2(-/-) and IRF9(-/-) MEFs that induction of ADAR1 p150 occurs by STAT2- and IRF9-dependent signaling that is enhanced by, but not obligatorily dependent upon, STAT1. Chromatin immunoprecipitation analysis demonstrated STAT2 at the PA promoter in IFN-treated Stat1(-/-) cells, whereas IFN-treated wild-type cells showed both STAT1 and STAT2 bound at PA. By contrast, with human 2fTGH cells and mutants U3A or U6A, ADAR1 induction by IFN was dependent upon both STAT1 and STAT2. These results suggest that transcriptional activation of Adar1 by IFN occurs in the absence of STAT1 by a non-canonical STAT2-dependent pathway in mouse but not human cells.
Assuntos
Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Regulação da Expressão Gênica , Interferon Tipo I/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Interferon Tipo I/farmacologia , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT2/genética , Ativação TranscricionalRESUMO
UNLABELLED: Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE: Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity. Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant.
Assuntos
Adenosina Desaminase/genética , Vírus do Sarampo/genética , Sarampo/enzimologia , Mutação , RNA Interferente Pequeno/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/genética , Adenosina Desaminase/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Sarampo/genética , Sarampo/virologia , Vírus do Sarampo/metabolismo , Estabilidade Proteica , Edição de RNA , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
Human adenovirus type 12 (HAdV-12) displays a relatively low virulence and slow replication in cultured human cells, which is manifested by premature death of HAdV-12-infected cells. Whereas HAdV-2 induction of IFN-ß expression was transient, HAdV-12-infected cells maintained high levels of IFN-ß expression, protein kinase R (PKR) activation and eIF-2α phosphorylation throughout the infectious cycle. The importance of the IFN-inducible PKR kinase in restriction of HAdV-12 was supported by the enhanced growth of the virus following PKR knockdown in HeLa cells. Ectopic expression of HAdV-2 VA RNAI increased HAdV-12 hexon protein expression, suggesting that insufficient VA RNA expression contributes to the restricted growth of HAdV-12. Although some adenovirus species are known to persist in human lymphoid tissues, HAdV12 has so far not been found. Thus, it is possible that the inability of HAdV12 to evade the INF response may have implications for the virus to establish long-lasting or persistent infections.
Assuntos
Adenovírus Humanos/crescimento & desenvolvimento , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Replicação Viral/fisiologia , eIF-2 Quinase/metabolismo , Adenovírus Humanos/classificação , Adenovírus Humanos/enzimologia , Morte Celular , Linhagem Celular , Linhagem Celular Tumoral , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Interferons , Fosforilação , eIF-2 Quinase/genéticaRESUMO
Adenosine deaminase acting on RNA1 (ADAR1) catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in regions of RNA with double-stranded (ds) character. This process is known as A-to-I RNA editing. Alternative promoters drive the expression of the Adar1 gene and alternative splicing gives rise to transcripts that encode 2 ADAR1 protein size isoforms. ADAR1 p150 is an interferon (IFN)-inducible dsRNA adenosine deaminase found in the cytoplasm and nucleus, whereas ADAR1 p110 is constitutively expressed and nuclear in localization. Dependent on the duplex structure of the dsRNA substrate, deamination of adenosine by ADAR can be either highly site-selective or nonspecific. A-to-I editing can alter the stability of RNA structures and the coding of RNA as I is read as G instead of A by ribosomes during mRNA translation and by polymerases during RNA replication. A-to-I editing is of broad physiologic significance. Both the production and the action of IFNs, and hence the subsequent interaction of viruses with their hosts, are among the processes affected by A-to-I editing.