RESUMO
Glycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammals use RNA as a third scaffold for glycosylation. Using a battery of chemical and biochemical approaches, we found that conserved small noncoding RNAs bear sialylated glycans. These "glycoRNAs" were present in multiple cell types and mammalian species, in cultured cells, and in vivo. GlycoRNA assembly depends on canonical N-glycan biosynthetic machinery and results in structures enriched in sialic acid and fucose. Analysis of living cells revealed that the majority of glycoRNAs were present on the cell surface and can interact with anti-dsRNA antibodies and members of the Siglec receptor family. Collectively, these findings suggest the existence of a direct interface between RNA biology and glycobiology, and an expanded role for RNA in extracellular biology.
Assuntos
Membrana Celular/metabolismo , Polissacarídeos/metabolismo , RNA/metabolismo , Animais , Anticorpos/metabolismo , Sequência de Bases , Vias Biossintéticas , Linhagem Celular , Sobrevivência Celular , Humanos , Espectrometria de Massas , Ácido N-Acetilneuramínico/metabolismo , Poliadenilação , Polissacarídeos/química , RNA/química , RNA/genética , RNA não Traduzido/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Coloração e RotulagemRESUMO
Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.
Assuntos
Dicistroviridae/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/virologia , Proteínas de Ligação ao GTP/metabolismo , Hepatócitos/virologia , Vírus de Insetos/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular Tumoral , Drosophila melanogaster/metabolismo , Hepacivirus/metabolismo , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Receptores de Quinase C Ativada , Sequências Reguladoras de Ácido Ribonucleico , Replicação ViralRESUMO
The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.
Assuntos
Hepatite D , Vírus Delta da Hepatite , Camundongos , Animais , Humanos , Roedores , Vírus da Hepatite B/genética , Serpentes , Replicação Viral , RNA Viral/genéticaRESUMO
As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats.
Assuntos
Vírus de RNA/genética , RNA Mensageiro/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética , Viroses/genética , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Ligação Proteica , Biossíntese de Proteínas , Vírus de RNA/crescimento & desenvolvimento , Vírus de RNA/patogenicidade , RNA Mensageiro/imunologia , RNA Viral/imunologia , Proteínas de Ligação a RNA/imunologia , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais , Proteínas Virais/metabolismo , Montagem de Vírus/genética , Viroses/imunologia , Viroses/patologia , Viroses/virologiaRESUMO
Many viruses interface with the autophagy pathway, a highly conserved process for recycling cellular components. For three viral infections in which autophagy constituents are proviral (poliovirus, dengue, and Zika), we developed a panel of knockouts (KOs) of autophagy-related genes to test which components of the canonical pathway are utilized. We discovered that each virus uses a distinct set of initiation components; however, all three viruses utilize autophagy-related gene 9 (ATG9), a lipid scavenging protein, and LC3 (light-chain 3), which is involved in membrane curvature. These results show that viruses use noncanonical routes for membrane sculpting and LC3 recruitment. By measuring viral RNA abundance, we also found that poliovirus utilizes these autophagy components for intracellular growth, while dengue and Zika virus only use autophagy components for post-RNA replication processes. Comparing how RNA viruses manipulate the autophagy pathway reveals new noncanonical autophagy routes, explains the exacerbation of disease by starvation, and uncovers common targets for antiviral drugs.
Assuntos
Autofagia/genética , Vírus de RNA/genética , Vírus de RNA/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Células HeLa , Humanos , Poliomielite/virologia , Poliovirus/genética , Poliovirus/fisiologia , Vírus de RNA/metabolismo , RNA Viral , Viroses/genética , Replicação Viral , Zika virus/genética , Zika virus/fisiologia , Infecção por Zika virus/virologiaRESUMO
The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.
Assuntos
Sistemas CRISPR-Cas/genética , Vírus da Dengue/fisiologia , Genoma Humano/genética , Hepacivirus/fisiologia , Fatores Celulares Derivados do Hospedeiro/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Dengue/genética , Vírus da Dengue/crescimento & desenvolvimento , Descoberta de Drogas , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Flavina-Adenina Dinucleotídeo/biossíntese , Flavina-Adenina Dinucleotídeo/metabolismo , Infecções por Flavivirus/genética , Infecções por Flavivirus/virologia , Glicosilação , Hexosiltransferases/deficiência , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Terapia de Alvo Molecular , Ligação Proteica , Sinais Direcionadores de Proteínas , Proteínas de Ligação a RNA/genética , Receptores Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Zika virus/metabolismoRESUMO
Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by â¼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to â¼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by â¼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by â¼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.
Assuntos
Sistemas CRISPR-Cas , DNA Helicases/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Dependovirus/genética , Edição de Genes , Células-Tronco Hematopoéticas/metabolismo , RecQ Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vetores Genéticos , Células HeLa , Células-Tronco Hematopoéticas/citologia , Recombinação Homóloga , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
RNA-protein interactions play numerous roles in cellular function and disease. Here we describe RNA-protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA-protein interactions in living cells on a timescale as short as 1 min.
Assuntos
Biotina/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Proteínas Virais/metabolismo , Zika virus/metabolismo , Bacillus subtilis/metabolismo , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , Neurônios/citologia , Neurônios/metabolismo , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Zika virus/genéticaRESUMO
Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.
Assuntos
Nucleotídeos de Citosina/genética , Genoma Viral , Hepacivirus/genética , Hepatite C/virologia , MicroRNAs/metabolismo , Mutação , RNA Viral/genética , Sítios de Ligação , Hepatite C/genética , Hepatite C/metabolismo , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/genética , Replicação ViralRESUMO
Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an â¼800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation.
Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Corantes Fluorescentes/química , Imagem Individual de Molécula/métodos , Análise Espectral/métodos , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/genética , Hepacivirus/genética , Humanos , Sítios Internos de Entrada Ribossomal/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Reprodutibilidade dos Testes , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
Since the initial description of Toll receptors in Drosophila and their mammalian counterparts Toll-like receptors (TLRs), numerous fundamental and applied studies have explored their crucial role as sensors of pathogen-associated molecular patterns (PAMPs). Among the ten human TLRs, TLR4 is particularly well known for its ability to detect lipopolysaccharides (LPS), a component of the Gram-negative bacterial cell wall. In addition to its archetypal functions, TLR4 is also a versatile virus sensor. This review provides a background on the discovery of TLR4 and how this knowledge laid a foundation for characterization of its diverse roles in antiviral responses, examined through genetic, biochemical, structural, and immunological approaches. These advances have led to a deeper understanding of the molecular functions that enable TLR4 to orchestrate multi-nodal control by professional antigen-presenting cells (APCs) to initiate appropriate and regulated antiviral immune responses.
RESUMO
Viral neuroinfections represent a major health burden for which the development of antivirals is needed. Antiviral compounds that target the consequences of a brain infection (symptomatic treatment) rather than the cause (direct-acting antivirals) constitute a promising mitigation strategy that requires to be investigated in relevant models. However, physiological surrogates mimicking an adult human cortex are lacking, limiting our understanding of the mechanisms associated with viro-induced neurological disorders. Here, we optimized the Organotypic culture of Post-mortem Adult human cortical Brain explants (OPAB) as a preclinical platform for Artificial Intelligence (AI)-driven antiviral studies. OPAB shows robust viability over weeks, well-preserved 3D cytoarchitecture, viral permissiveness, and spontaneous local field potential (LFP). Using LFP as a surrogate for neurohealth, we developed a machine learning framework to predict with high confidence the infection status of OPAB. As a proof-of-concept, we showed that antiviral-treated OPAB could partially restore LFP-based electrical activity of infected OPAB in a donor-dependent manner. Together, we propose OPAB as a physiologically relevant and versatile model to study neuroinfections and beyond, providing a platform for preclinical drug discovery.
Assuntos
Antivirais , Hepatite C Crônica , Humanos , Antivirais/farmacologia , Inteligência Artificial , Sistemas Microfisiológicos , EncéfaloRESUMO
Hepatitis delta virus (HDV), a satellite of hepatitis B virus (HBV), possesses the smallest viral genome known to infect animals. HDV needs HBV surface protein for secretion and entry into target liver cells. However, HBV is dispensable for HDV genome amplification, as it relies almost exclusively on cellular host factors for replication. HBV/HDV co-infections affect over 12 million people worldwide and constitute the most severe form of viral hepatitis. Co-infected individuals are at higher risk of developing liver cirrhosis and hepatocellular carcinoma compared to HBV mono-infected patients. Bulevirtide, an entry inhibitor, was conditionally approved in July 2020 in the European Union for adult patients with chronic hepatitis delta (CHD) and compensated liver disease. There are several drugs in development, including lonafarnib and interferon lambda, with different modes of action. In this review, we detail our current fundamental knowledge of HDV lifecycle and review antiviral treatments under development against this virus, outlining their respective mechanisms-of-action. Finally, we describe the antiviral effect these compounds are showing in ongoing clinical trials, discussing their promise and potential pitfalls for managing HDV infected patients.
Assuntos
Hepatite B , Hepatite D , Animais , Vírus Delta da Hepatite , Hepatite D/tratamento farmacológico , Antivirais/farmacologia , Antivirais/uso terapêutico , Vírus da Hepatite B/genéticaRESUMO
Chronic HBV infection is a major cause of liver disease and cancer worldwide. Approaches for cure are lacking, and the knowledge of virus-host interactions is still limited. Here, we perform a genome-wide gain-of-function screen using a poorly permissive hepatoma cell line to uncover host factors enhancing HBV infection. Validation studies in primary human hepatocytes identified CDKN2C as an important host factor for HBV replication. CDKN2C is overexpressed in highly permissive cells and HBV-infected patients. Mechanistic studies show a role for CDKN2C in inducing cell cycle G1 arrest through inhibition of CDK4/6 associated with the upregulation of HBV transcription enhancers. A correlation between CDKN2C expression and disease progression in HBV-infected patients suggests a role in HBV-induced liver disease. Taken together, we identify a previously undiscovered clinically relevant HBV host factor, allowing the development of improved infectious model systems for drug discovery and the study of the HBV life cycle.
Assuntos
Inibidor de Quinase Dependente de Ciclina p18/genética , Mutação com Ganho de Função , Testes Genéticos/métodos , Estudo de Associação Genômica Ampla/métodos , Hepatite B/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Perfilação da Expressão Gênica/métodos , Células HEK293 , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Interações entre Hospedeiro e Microrganismos , Humanos , Estimativa de Kaplan-Meier , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Interferência de RNA , Replicação Viral/fisiologiaRESUMO
Animal cells have evolved dedicated molecular systems for sensing and delivering a coordinated response to viral threats. Our understanding of these pathways is almost entirely defined by studies in humans or model organisms like mice, fruit flies and worms. However, new genomic and functional data from organisms such as sponges, anemones and mollusks are helping redefine our understanding of these immune systems and their evolution. In this review, we will discuss our current knowledge of the innate immune pathways involved in sensing, signaling and inducing genes to counter viral infections in vertebrate animals. We will then focus on some central conserved players of this response including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and cGAS-STING, attempting to put their evolution into perspective. To conclude, we will reflect on the arms race that exists between viruses and their animal hosts, illustrated by the dynamic evolution and diversification of innate immune pathways. These concepts are not only important to understand virus-host interactions in general but may also be relevant for the development of novel curative approaches against human disease.
Assuntos
Evolução Biológica , Imunidade Inata , Viroses/imunologia , Animais , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/imunologia , Humanos , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Viroses/genética , Fenômenos Fisiológicos Virais , Vírus/genéticaRESUMO
Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.
Assuntos
Infecções por Flavivirus/virologia , Flavivirus/genética , Flavivirus/fisiologia , RNA Viral/genética , Sistemas CRISPR-Cas , Proteínas de Transporte , Linhagem Celular , Vírus da Dengue/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Flavivirus/patogenicidade , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/genética , Humanos , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Replicação Viral , Zika virus/genéticaRESUMO
Viruses depend on their hosts to complete their replication cycles; they exploit cellular receptors for entry and hijack cellular functions to replicate their genome, assemble progeny virions and spread. Recently, genome-scale CRISPR-Cas screens have been used to identify host factors that are required for virus replication, including the replication of clinically relevant viruses such as Zika virus, West Nile virus, dengue virus and hepatitis C virus. In this Review, we discuss the technical aspects of genome-scale knockout screens using CRISPR-Cas technology, and we compare these screens with alternative genetic screening technologies. The relative ease of use and reproducibility of CRISPR-Cas make it a powerful tool for probing virus-host interactions and for identifying new antiviral targets.
Assuntos
Sistemas CRISPR-Cas/genética , Vírus da Dengue/genética , Hepacivirus/genética , Ensaios de Triagem em Larga Escala/métodos , Vírus do Nilo Ocidental/genética , Zika virus/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas de Inativação de Genes , Humanos , Interferência de RNA , Replicação Viral/genéticaRESUMO
Receptor for Activated protein C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism Drosophila melanogaster, we recently showed that RACK1 is required at the ribosome for internal ribosome entry site (IRES)-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in Drosophila S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition (DDA and DIA, respectively) and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method, both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for Drosophila and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation, and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus (CrPV), an IRES-containing virus.
Assuntos
Dicistroviridae , Proteínas de Drosophila , Redes Reguladoras de Genes , Sítios Internos de Entrada Ribossomal , Modelos Genéticos , Receptores de Quinase C Ativada , Proteínas Virais , Animais , Linhagem Celular , Dicistroviridae/genética , Dicistroviridae/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Biossíntese de Proteínas/genética , Receptores de Quinase C Ativada/genética , Receptores de Quinase C Ativada/metabolismo , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses.