RESUMO
SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/virologia , Imunidade Inata/genética , Pandemias , SARS-CoV-2/genéticaRESUMO
Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
Assuntos
COVID-19 , RNA Viral , Humanos , COVID-19/metabolismo , Endonucleases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação ViralRESUMO
The Coronaviridae are a family of viruses that cause disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E), and glycosaminoglycans (for OC43). Additionally, we identified phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol kinases and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle and the development of host-directed therapies.
Assuntos
COVID-19/genética , Infecções por Coronavirus/genética , Coronavirus/fisiologia , Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Células A549 , Animais , Vias Biossintéticas/efeitos dos fármacos , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Colesterol/biossíntese , Colesterol/metabolismo , Análise por Conglomerados , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resfriado Comum/genética , Resfriado Comum/virologia , Coronavirus/classificação , Infecções por Coronavirus/virologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos , Fosfatidilinositóis/biossíntese , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação ViralRESUMO
Viruses are a constant threat to global health as highlighted by the current COVID-19 pandemic. Currently, lack of data underlying how the human host interacts with viruses, including the SARS-CoV-2 virus, limits effective therapeutic intervention. We introduce Viral-Track, a computational method that globally scans unmapped single-cell RNA sequencing (scRNA-seq) data for the presence of viral RNA, enabling transcriptional cell sorting of infected versus bystander cells. We demonstrate the sensitivity and specificity of Viral-Track to systematically detect viruses from multiple models of infection, including hepatitis B virus, in an unsupervised manner. Applying Viral-Track to bronchoalveloar-lavage samples from severe and mild COVID-19 patients reveals a dramatic impact of the virus on the immune system of severe patients compared to mild cases. Viral-Track detects an unexpected co-infection of the human metapneumovirus, present mainly in monocytes perturbed in type-I interferon (IFN)-signaling. Viral-Track provides a robust technology for dissecting the mechanisms of viral-infection and pathology.
Assuntos
Infecções por Coronavirus/fisiopatologia , Interações Hospedeiro-Patógeno , Pneumonia Viral/fisiopatologia , Software , Animais , Betacoronavirus/isolamento & purificação , COVID-19 , Coinfecção/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Interferons/imunologia , Pulmão/patologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SARS-CoV-2 , Sensibilidade e Especificidade , Análise de Sequência de RNA , Índice de Gravidade de Doença , Análise de Célula ÚnicaRESUMO
Viral pandemics, such as the one caused by SARS-CoV-2, pose an imminent threat to humanity. Because of its recent emergence, there is a paucity of information regarding viral behavior and host response following SARS-CoV-2 infection. Here we offer an in-depth analysis of the transcriptional response to SARS-CoV-2 compared with other respiratory viruses. Cell and animal models of SARS-CoV-2 infection, in addition to transcriptional and serum profiling of COVID-19 patients, consistently revealed a unique and inappropriate inflammatory response. This response is defined by low levels of type I and III interferons juxtaposed to elevated chemokines and high expression of IL-6. We propose that reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19.
Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Vírus de RNA/imunologia , Animais , COVID-19 , Células Cultivadas , Quimiocinas/genética , Quimiocinas/imunologia , Infecções por Coronavirus/genética , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Inflamação/virologia , Interferons/genética , Interferons/imunologia , Pandemias , Pneumonia Viral/genética , Vírus de RNA/classificação , SARS-CoV-2 , Transcrição GênicaRESUMO
Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.
Assuntos
Proteínas de Transporte , Proteínas de Ligação a DNA , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/metabolismo , Fatores de Transcrição , Proteínas Virais , Replicação Viral/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/patologia , Humanos , Mapeamento de Interação de Proteínas , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.
Assuntos
Infecções por HIV , Ácidos Nucleicos , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Linfócitos T CD4-Positivos , Vírus de DNA , Terapia de Imunossupressão , Macaca mulatta , Macrófagos , Vírus da Imunodeficiência Símia/fisiologia , Carga ViralRESUMO
Gut microbial dysbioses are linked to aberrant immune responses, which are often accompanied by abnormal production of inflammatory cytokines. As part of the Human Functional Genomics Project (HFGP), we investigate how differences in composition and function of gut microbial communities may contribute to inter-individual variation in cytokine responses to microbial stimulations in healthy humans. We observe microbiome-cytokine interaction patterns that are stimulus specific, cytokine specific, and cytokine and stimulus specific. Validation of two predicted host-microbial interactions reveal that TNFα and IFNγ production are associated with specific microbial metabolic pathways: palmitoleic acid metabolism and tryptophan degradation to tryptophol. Besides providing a resource of predicted microbially derived mediators that influence immune phenotypes in response to common microorganisms, these data can help to define principles for understanding disease susceptibility. The three HFGP studies presented in this issue lay the groundwork for further studies aimed at understanding the interplay between microbial, genetic, and environmental factors in the regulation of the immune response in humans. PAPERCLIP.
Assuntos
Citocinas/imunologia , Microbioma Gastrointestinal , Inflamação/imunologia , Microbiota , Adolescente , Adulto , Idoso , Bactérias/classificação , Bactérias/imunologia , Sangue/imunologia , Disbiose/imunologia , Disbiose/microbiologia , Fezes/microbiologia , Feminino , Fungos/classificação , Fungos/imunologia , Interação Gene-Ambiente , Projeto Genoma Humano , Humanos , Infecções/imunologia , Infecções/microbiologia , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Nutrient sensing and damage sensing are two fundamental processes in living organisms. While hyperglycemia is frequently linked to diabetes-related vulnerability to microbial infection, how body glucose levels affect innate immune responses to microbial invasion is not fully understood. Here, we surprisingly found that viral infection led to a rapid and dramatic decrease in blood glucose levels in rodents, leading to robust AMPK activation. AMPK, once activated, directly phosphorylates TBK1 at S511, which triggers IRF3 recruitment and the assembly of MAVS or STING signalosomes. Consistently, ablation or inhibition of AMPK, knockin of TBK1-S511A, or increased glucose levels compromised nucleic acid sensing, while boosting AMPK-TBK1 cascade by AICAR or TBK1-S511E knockin improves antiviral immunity substantially in various animal models. Thus, we identify TBK1 as an AMPK substrate, reveal the molecular mechanism coupling a dual sensing of glucose and nuclei acids, and report its physiological necessity in antiviral defense.
Assuntos
Proteínas Quinases Ativadas por AMP , Ácidos Nucleicos , Animais , Proteínas Quinases Ativadas por AMP/genética , Imunidade Inata , Antivirais , GlucoseRESUMO
N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.
Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The ChvG-ChvI two-component system is conserved among multiple Alphaproteobacteria. ChvG is a canonical two-component system sensor kinase with a single large periplasmic loop. Active ChvG directs phosphotransfer to its cognate response regulator ChvI, which controls transcription of target genes. In many alphaproteobacteria, ChvG is regulated by a third component, a periplasmic protein called ExoR, that maintains ChvG in an inactive state through direct interaction. Acidic pH stimulates proteolysis of ExoR, unfettering ChvG-ChvI to control its regulatory targets. Activated ChvI among different alphaproteobacteria controls a broad range of cellular processes, including symbiosis and virulence, exopolysaccharide production, biofilm formation, motility, type VI secretion, cellular metabolism, envelope composition, and growth. Low pH is a virulence signal in Agrobacterium tumefaciens, but in other systems, conditions that cause envelope stress may also generally activate ChvG-ChvI. There is mounting evidence that these regulators influence diverse aspects of bacterial physiology, including but not limited to host interactions.
Assuntos
Agrobacterium tumefaciens , Proteínas de Bactérias , Proteínas de Bactérias/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Transdução de Sinais/genética , SimbioseRESUMO
Bacteriophage research has been vital to fundamental aspects of modern biology. Advances in metagenomics have revealed treasure troves of new and uncharacterized bacteriophages ('phages') that are not yet understood. However, our ability to find new phages has outpaced our understanding of how sequence encodes function in phages. Traditional approaches for characterizing phages are limited in scale and face hurdles in determining how changes in sequence drive function. We describe powerful emerging technologies that can be used to clarify sequence-function relationships in phages through high-throughput genome engineering. Using these approaches, up to 105 variants can be characterized through pooled selection experiments and deep sequencing. We describe caveats when using these tools and provide examples of basic science and engineering goals that are pursuable using these approaches.
Assuntos
Bacteriófagos , Bacteriófagos/genética , Genoma ViralRESUMO
Herpesviruses are DNA viruses and the cause of diseases ranging from mild skin conditions to severe brain diseases. Mammalian antiviral host defense comprises an array of mechanisms, including restriction factors (RFs), which block specific steps in viral replication cycles. In recent years, knowledge of RFs that contribute to controlling herpesvirus infections has expanded significantly, along with a new understanding of viral evasion mechanisms and disease pathogenesis. By integrating findings from human genetics, murine models, and cellular studies, this review provides a current view of RF control of herpesvirus infections. We also explore the regulation of RF expression, discuss the roles of RFs in diseases, and point towards their growing potential as candidate therapeutic targets.
RESUMO
Organic carbon availability in soil is crucial for shaping microbial communities, yet, uncertainties persist concerning microbial adaptations to carbon levels and the ensuing ecological and evolutionary consequences. We investigated organic carbon metabolism, antibiotic resistance, and virus-host interactions in soils subjected to 40 y of chemical and organic fertilization that led to contrasting carbon availability: carbon-poor and carbon-rich soils, respectively. Carbon-poor soils drove the enrichment of putative genes involved in organic matter decomposition and exhibited specialization in utilizing complex organic compounds, reflecting scramble competition. This specialization confers a competitive advantage of microbial communities in carbon-poor soils but reduces their buffering capacity in terms of organic carbon metabolisms, making them more vulnerable to environmental fluctuations. Additionally, in carbon-poor soils, viral auxiliary metabolic genes linked to organic carbon metabolism increased host competitiveness and environmental adaptability through a strategy akin to "piggyback the winner." Furthermore, putative antibiotic resistance genes, particularly in low-abundance drug categories, were enriched in carbon-poor soils as an evolutionary consequence of chemical warfare (i.e., interference competition). This raises concerns about the potential dissemination of antibiotic resistance from conventional agriculture that relies on chemical-only fertilization. Consequently, carbon starvation resulting from long-term chemical-only fertilization increases microbial adaptations to competition, underscoring the importance of implementing sustainable agricultural practices to mitigate the emergence and spread of antimicrobial resistance and to increase soil carbon storage.
Assuntos
Carbono , Solo , Solo/química , Carbono/metabolismo , Agricultura/métodos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Microbiologia do SoloRESUMO
RNA viruses interact with a wide range of cellular RNA-binding proteins (RBPs) during their life cycle. The prevalence of these host-virus interactions has been highlighted by new methods that elucidate the composition of viral ribonucleoproteins (vRNPs). Applied to 11 viruses so far, these approaches have revealed hundreds of cellular RBPs that interact with viral (v)RNA in infected cells. However, consistency across methods is limited, raising questions about methodological considerations when designing and interpreting these studies. Here, we discuss these caveats and, through comparing available vRNA interactomes, describe RBPs that are consistently identified as vRNP components and outline their potential roles in infection. In summary, these novel approaches have uncovered a new universe of host-virus interactions holding great therapeutic potential.
Assuntos
Proteoma , RNA Viral , Comunicação Celular , Interações entre Hospedeiro e Microrganismos , Interações Hospedeiro-Patógeno , Proteoma/metabolismo , RNA Viral/genética , Ribonucleoproteínas/metabolismoRESUMO
The mature HIV-1 capsid protects the viral genome and interacts with host proteins to travel from the cell periphery into the nucleus. To achieve this, the capsid protein, CA, constructs conical capsids from a lattice of hexamers and pentamers, and engages in and then relinquishes multiple interactions with cellular proteins in an orchestrated fashion. Cellular host factors including Nup153, CPSF6, and Sec24C engage the same pocket within CA hexamers. How CA assembles pentamers and hexamers of different curvatures, how CA oligomerization states or curvature might modulate host-protein interactions, and how binding of multiple cofactors to a single site is coordinated, all remain to be elucidated. Here, using single-particle cryoEM, we have determined the structure of the mature HIV-1 CA pentamer and hexamer from conical CA-IP6 polyhedra to ~3 Å resolution. We also determined structures of hexamers in the context of multiple lattice curvatures and number of pentamer contacts. Comparison of these structures, bound or not to host protein peptides, revealed two structural switches within HIV-1 CA that modulate peptide binding according to CA lattice curvature and whether CA is hexameric or pentameric. These observations suggest that the conical HIV-1 capsid has different host-protein binding properties at different positions on its surface, which may facilitate cell entry and represent an evolutionary advantage of conical morphology.
Assuntos
Capsídeo , HIV-1 , Capsídeo/metabolismo , Proteínas do Capsídeo/química , HIV-1/genética , Ligação Proteica , Citoplasma/metabolismoRESUMO
The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, and exert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.
Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Prolina/metabolismo , Tirosina/metabolismo , Proteínas de Transporte , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Replicação ViralRESUMO
The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.
Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Regulação Viral da Expressão Gênica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/fisiologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Cromatografia em Gel , Proteases Semelhantes à Papaína de Coronavírus/química , Cristalografia por Raios X , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Viral/genética , Proteínas de Ligação a RNA/química , RNA Polimerase Dependente de RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Subunidades Ribossômicas/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , SARS-CoV-2/genética , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Difração de Raios XRESUMO
The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.
Assuntos
COVID-19 , Proteínas do Envelope Viral , Animais , Humanos , Proteínas do Envelope Viral/metabolismo , Proteínas Viroporinas/metabolismo , COVID-19/metabolismo , Prótons , Pandemias , SARS-CoV-2/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Mamíferos/metabolismoRESUMO
The host-virus interactome is increasingly recognized as an important research field to discover new therapeutic targets to treat influenza. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify new pro- and antiviral host factors of the influenza A virus. However, at present, a comprehensive summary of the results is lacking. We performed a systematic review of all reported CRISPR studies in this field in combination with a meta-analysis using the algorithm of meta-analysis by information content (MAIC). Two ranked gene lists were generated based on evidence in 15 proviral and 4 antiviral screens. Enriched pathways in the proviral MAIC results were compared to those of a prior array-based RNA interference (RNAi) meta-analysis. The top 50 proviral MAIC list contained genes whose role requires further elucidation, such as the endosomal ion channel TPCN1 and the kinase WEE1. Moreover, MAIC indicated that ALYREF, a component of the transcription export complex, has antiviral properties, whereas former knockdown experiments attributed a proviral role to this host factor. CRISPR-Cas-pooled screens displayed a bias toward early-replication events, whereas the prior RNAi meta-analysis covered early and late-stage events. RNAi screens led to the identification of a larger fraction of essential genes than CRISPR screens. In summary, the MAIC algorithm points toward the importance of several less well-known pathways in host-influenza virus interactions that merit further investigation. The results from this meta-analysis of CRISPR screens in influenza A virus infection may help guide future research efforts to develop host-directed anti-influenza drugs. IMPORTANCE: Viruses rely on host factors for their replication, whereas the host cell has evolved virus restriction factors. These factors represent potential targets for host-oriented antiviral therapies. Multiple pooled genome-wide CRISPR-Cas screens have been reported to identify pro- and antiviral host factors in the context of influenza virus infection. We performed a comprehensive analysis of the outcome of these screens based on the publicly available gene lists, using the recently developed algorithm meta-analysis by information content (MAIC). MAIC allows the systematic integration of ranked and unranked gene lists into a final ranked gene list. This approach highlighted poorly characterized host factors and pathways with evidence from multiple screens, such as the vesicle docking and lipid metabolism pathways, which merit further exploration.