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1.
EMBO Rep ; 23(11): e54061, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36161446

RESUMO

Genome-wide screens are powerful approaches to unravel regulators of viral infections. Here, a CRISPR screen identifies the RNA helicase DDX42 as an intrinsic antiviral inhibitor of HIV-1. Depletion of endogenous DDX42 increases HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibits HIV-1 infection, whereas expression of a dominant-negative mutant increases infection. Importantly, DDX42 also restricts LINE-1 retrotransposition and infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 does not impact the replication of several negative-strand RNA viruses, arguing against an unspecific effect on target cells, which is confirmed by RNA-seq analysis. Proximity ligation assays show DDX42 in the vicinity of viral elements, and cross-linking RNA immunoprecipitation confirms a specific interaction of DDX42 with RNAs from sensitive viruses. Moreover, recombinant DDX42 inhibits HIV-1 reverse transcription in vitro. Together, our data strongly suggest a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Our results identify DDX42 as an intrinsic viral inhibitor, opening new perspectives to target the life cycle of numerous RNA viruses.


Assuntos
RNA Helicases DEAD-box , HIV-1 , Vírus de RNA de Cadeia Positiva , Replicação Viral , Humanos , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , HIV-1/fisiologia , Vírus de RNA de Cadeia Positiva/fisiologia , SARS-CoV-2/fisiologia
2.
Proc Natl Acad Sci U S A ; 119(39): e2204624119, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36074824

RESUMO

The high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, postexposure TIP dose lowers SARS-CoV-2 nasal shedding, and at 5 days postinfection, infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Cohoused "contact" animals exposed to infected, TIP-treated animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals cohoused with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission.


Assuntos
COVID-19 , RNA Mensageiro , RNA Interferente Pequeno , SARS-CoV-2 , Eliminação de Partículas Virais , Animais , COVID-19/terapia , COVID-19/transmissão , Cricetinae , RNA Mensageiro/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , SARS-CoV-2/genética , SARS-CoV-2/fisiologia
3.
Proc Natl Acad Sci U S A ; 119(38): e2209514119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36048924

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein-catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.


Assuntos
COVID-19 , Cavidade Nasal , SARS-CoV-2 , Serina Endopeptidases , Internalização do Vírus , COVID-19/virologia , Furina/genética , Furina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cavidade Nasal/química , Cavidade Nasal/virologia , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
Nat Commun ; 13(1): 5196, 2022 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-36057636

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes COVID-19, produces polyproteins 1a and 1ab that contain, respectively, 11 or 16 non-structural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for viral assembly and maturation. Using C-terminally substituted Mpro chimeras, we have determined X-ray crystallographic structures of Mpro in complex with 10 of its 11 viral cleavage sites, bound at full occupancy intermolecularly in trans, within the active site of either the native enzyme and/or a catalytic mutant (C145A). Capture of both acyl-enzyme intermediate and product-like complex forms of a P2(Leu) substrate in the native active site provides direct comparative characterization of these mechanistic steps as well as further informs the basis for enhanced product release of Mpro's own unique C-terminal P2(Phe) cleavage site to prevent autoinhibition. We characterize the underlying noncovalent interactions governing binding and specificity for this diverse set of substrates, showing remarkable plasticity for subsites beyond the anchoring P1(Gln)-P2(Leu/Val/Phe), representing together a near complete analysis of a multiprocessing viral protease. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for antiviral therapeutic development.


Assuntos
COVID-19 , Proteases 3C de Coronavírus/metabolismo , Poliproteínas , SARS-CoV-2/fisiologia , Cisteína Endopeptidases/metabolismo , Humanos , Peptídeo Hidrolases , Poliproteínas/química , Proteínas Virais/química , Raios X
5.
Emerg Microbes Infect ; 11(1): 2160-2175, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36000328

RESUMO

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.


Assuntos
COVID-19 , Pulmão , Infecções por Orthomyxoviridae , Animais , Antivirais/farmacologia , COVID-19/patologia , Fluoxetina/farmacologia , Humanos , Vírus da Influenza A/fisiologia , Influenza Humana/patologia , Interferons , Pulmão/virologia , Camundongos , Infecções por Orthomyxoviridae/patologia , SARS-CoV-2/fisiologia , Técnicas de Cultura de Tecidos , Replicação Viral
6.
mBio ; 13(4): e0194422, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35938726

RESUMO

The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.


Assuntos
COVID-19 , Mucosa Nasal , Organoides , SARS-CoV-2 , COVID-19/virologia , Humanos , Mucosa Nasal/virologia , Organoides/virologia , SARS-CoV-2/classificação , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Técnicas de Cultura de Tecidos
7.
Proc Natl Acad Sci U S A ; 119(30): e2122236119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35858406

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.


Assuntos
Astrócitos , Córtex Cerebral , SARS-CoV-2 , Tropismo Viral , Enzima de Conversão de Angiotensina 2/metabolismo , Astrócitos/enzimologia , Astrócitos/virologia , Córtex Cerebral/virologia , Humanos , Organoides/virologia , Cultura Primária de Células , SARS-CoV-2/fisiologia
8.
BMC Genomics ; 23(1): 497, 2022 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-35804296

RESUMO

BACKGROUND: Emerging infectious disease-causing RNA viruses, such as the SARS-CoV-2 and Ebola viruses, are thought to rely on bats as natural reservoir hosts. Since these zoonotic viruses pose a great threat to humans, it is important to characterize the bat genome from multiple perspectives. Unsupervised machine learning methods for extracting novel information from big sequence data without prior knowledge or particular models are highly desirable for obtaining unexpected insights. We previously established a batch-learning self-organizing map (BLSOM) of the oligonucleotide composition that reveals novel genome characteristics from big sequence data. RESULTS: In this study, using the oligonucleotide BLSOM, we conducted a comparative genomic study of humans and six bat species. BLSOM is an explainable-type machine learning algorithm that reveals the diagnostic oligonucleotides contributing to sequence clustering (self-organization). When unsupervised machine learning reveals unexpected and/or characteristic features, these features can be studied in more detail via the much simpler and more direct standard distribution map method. Based on this combined strategy, we identified the Mb-level enrichment of CG dinucleotide (Mb-level CpG islands) around the termini of bat long-scaffold sequences. In addition, a class of CG-containing oligonucleotides were enriched in the centromeric and pericentromeric regions of human chromosomes. Oligonucleotides longer than tetranucleotides often represent binding motifs for a wide variety of proteins (e.g., transcription factor binding sequences (TFBSs)). By analyzing the penta- and hexanucleotide composition, we observed the evident enrichment of a wide range of hexanucleotide TFBSs in centromeric and pericentromeric heterochromatin regions on all human chromosomes. CONCLUSION: Function of transcription factors (TFs) beyond their known regulation of gene expression (e.g., TF-mediated looping interactions between two different genomic regions) has received wide attention. The Mb-level TFBS and CpG islands are thought to be involved in the large-scale nuclear organization, such as centromere and telomere clustering. TFBSs, which are enriched in centromeric and pericentromeric heterochromatin regions, are thought to play an important role in the formation of nuclear 3D structures. Our machine learning-based analysis will help us to understand the differential features of nuclear 3D structures in the human and bat genomes.


Assuntos
COVID-19 , Quirópteros/genética , Genoma Humano/genética , SARS-CoV-2/fisiologia , Animais , COVID-19/transmissão , Quirópteros/virologia , Ilhas de CpG , Genômica/métodos , Heterocromatina/química , Heterocromatina/genética , Humanos , Conformação Molecular , Oligonucleotídeos/química , Aprendizado de Máquina não Supervisionado
9.
Proc Natl Acad Sci U S A ; 119(32): e2205690119, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35881779

RESUMO

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.


Assuntos
COVID-19 , Furina , Proteólise , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Motivos de Aminoácidos/genética , Animais , COVID-19/virologia , Chlorocebus aethiops , Furina/química , Humanos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Replicação Viral/genética
10.
ACS Infect Dis ; 8(8): 1533-1542, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35822715

RESUMO

SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5'-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 µM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.


Assuntos
Metiltransferases/metabolismo , RNA Helicases/metabolismo , SARS-CoV-2/química , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases , COVID-19/virologia , DNA de Cadeia Simples , Humanos , Metiltransferases/antagonistas & inibidores , Ácidos Nucleicos/metabolismo , RNA Helicases/antagonistas & inibidores , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/antagonistas & inibidores
11.
J Virol ; 96(15): e0075322, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35876526

RESUMO

Circulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the human population leads to further viral evolution. The new variants that arise during this evolution are more infectious. Our data suggest that newer variants have shifted from utilizing both cathepsin/endosome- and TMPRSS2-mediated entry mechanisms to rely on a TMPRSS2-dependent entry pathway. Accordingly, only the early lineages of SARS-CoV-2 are capable of infecting and forming syncytia in Vero/ACE2 cells which lack TMPRSS2 expression. The presence of an intact multibasic furin cleavage site (FCS) in the S protein was a key requirement for cell-to-cell fusion. Deletion of FCS makes SARS-CoV-2 more infectious in vitro but renders it incapable of syncytium formation. Cell-to-cell fusion likely represents an alternative means of virus spread and is resistant to the presence of high levels of neutralizing monoclonal antibodies (MAbs) and immune sera in the media. In this study, we also noted that cells infected with SARS-CoV-2 with an intact FCS or alphavirus replicon expressing S protein (VEErep/S) released high levels of free S1 subunit. The released S1 is capable of activating the TLR4 receptor and inducing a pro-inflammatory response. Thus, S1 activation of TLR4 may be an important contributor to SARS-CoV-2-induced COVID-19 disease and needs to be considered in the design of COVID mRNA vaccines. Lastly, a VEErep/S-replicon was shown to produce large amounts of infectious, syncytium-forming pseudoviruses and thus could represent alternative experimental system for screening inhibitors of virus entry and syncytium formation. IMPORTANCE The results of this study demonstrate that the late lineages of SARS-CoV-2 evolved to more efficient use of the TMPRSS2-mediated entry pathway and gradually lost an ability to employ the cathepsins/endosome-mediated entry. The acquisition of a furin cleavage site (FCS) by SARS-CoV-2-specific S protein made the virus a potent producer of syncytia. Their formation is also determined by expression of ACE2 and TMPRSS2 and is resistant to neutralizing human MAbs and immune sera. Syncytium formation appears to be an alternative means of infection spread following the development of an adaptive immune response. Cells infected with SARS-CoV-2 with an intact FCS secrete high levels of the S1 subunit. The released S1 demonstrates an ability to activate the TLR4 receptor and induce pro-inflammatory cytokines, which represent a hallmark of SARS-CoV-2 pathogenesis. Alphavirus replicons encoding SARS-CoV-2 S protein cause spreading, syncytium-forming infection, and they can be applied as an experimental tool for studying the mechanism of syncytium formation.


Assuntos
COVID-19 , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Evolução Molecular , Furina/metabolismo , Humanos , Soros Imunes , SARS-CoV-2/genética , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 4 Toll-Like , Internalização do Vírus
12.
Proc Natl Acad Sci U S A ; 119(30): e2123065119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35858407

RESUMO

SARS-CoV-2, the causative agent of the COVID-19 pandemic, undergoes continuous evolution, highlighting an urgent need for development of novel antiviral therapies. Here we show a quantitative mass spectrometry-based succinylproteomics analysis of SARS-CoV-2 infection in Caco-2 cells, revealing dramatic reshape of succinylation on host and viral proteins. SARS-CoV-2 infection promotes succinylation of several key enzymes in the TCA, leading to inhibition of cellular metabolic pathways. We demonstrated that host protein succinylation is regulated by viral nonstructural protein (NSP14) through interaction with sirtuin 5 (SIRT5); overexpressed SIRT5 can effectively inhibit virus replication. We found succinylation inhibitors possess significant antiviral effects. We also found that SARS-CoV-2 nucleocapsid and membrane proteins underwent succinylation modification, which was conserved in SARS-CoV-2 and its variants. Collectively, our results uncover a regulatory mechanism of host protein posttranslational modification and cellular pathways mediated by SARS-CoV-2, which may become antiviral drug targets against COVID-19.


Assuntos
Antivirais , COVID-19 , Interações Hospedeiro-Patógeno , Terapia de Alvo Molecular , Processamento de Proteína Pós-Traducional , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/metabolismo , COVID-19/virologia , Células CACO-2 , Exorribonucleases/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Sirtuínas/metabolismo , Succinatos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
13.
Structure ; 30(9): 1224-1232.e5, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35716662

RESUMO

Emerging new variants of SARS-CoV-2 and inevitable acquired drug resistance call for the continued search of new pharmacological targets to fight the potentially fatal infection. Here, we describe the mechanisms by which the E protein of SARS-CoV-2 hijacks the human transcriptional regulator BRD4. We found that SARS-CoV-2 E is acetylated in vivo and co-immunoprecipitates with BRD4 in human cells. Bromodomains (BDs) of BRD4 bind to the C-terminus of the E protein, acetylated by human acetyltransferase p300, whereas the ET domain of BRD4 recognizes the unmodified motif of the E protein. Inhibitors of BRD4 BDs, JQ1 or OTX015, decrease SARS-CoV-2 infectivity in lung bronchial epithelial cells, indicating that the acetyllysine binding function of BDs is necessary for the virus fitness and that BRD4 represents a potential anti-COVID-19 target. Our findings provide insight into molecular mechanisms that contribute to SARS-CoV-2 pathogenesis and shed light on a new strategy to block SARS-CoV-2 infection.


Assuntos
COVID-19 , Proteínas de Ciclo Celular/metabolismo , Proteínas do Envelope de Coronavírus/metabolismo , SARS-CoV-2/fisiologia , Fatores de Transcrição/metabolismo , COVID-19/virologia , Humanos , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios Proteicos
14.
PLoS One ; 17(6): e0270609, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35763527

RESUMO

Covid-19 progression shows sex-dependent features. It is hypothesized that a better Covid-19 survival rate in females can be attributed to the presence of higher 17ß-estradiol (E2) levels in women than in men. Virus SARS-CoV-2 is enabled to enter the cell with the use of angiotensin converting enzyme 2 (ACE2). The expression of several renin-angiotensin system components has been shown to exert a rhythmic pattern, and a role of the circadian system in their regulation has been implicated. Therefore, the aim of the study is to elucidate possible interference between E2 signalling and the circadian system in the regulation of the expression of ACE2 mRNA and functionally related molecules. E2 was administered at a dosage of 40 µg/kg/day for 7 days to male Wistar rats, and sampling of the lungs and colon was performed during a 24-h cycle. The daily pattern of expression of molecules facilitating SARS-CoV-2 entry into the cell, clock genes and E2 receptors was analysed. As a consequence of E2 administration, a rhythm in ACE2 and TMPRSS2 mRNA expression was observed in the lungs but not in the colon. ADAM17 mRNA expression showed a pronounced rhythmic pattern in both tissues that was not influenced by E2 treatment. ESR1 mRNA expression exerted a rhythmic pattern, which was diminished by E2 treatment. The influence of E2 administration on ESR2 and GPER1 mRNA expression was greater in the lungs than in the colon as a significant rhythm in ESR2 and GPER1 mRNA expression appeared only in the lungs after E2 treatment. E2 administration also increased the amplitude of bmal1 expression in the lungs, which implicates altered functioning of peripheral oscillators in response to E2 treatment. The daily pattern of components of the SARS-CoV-2 entrance pathway and their responsiveness to E2 should be considered in the timing of pharmacological therapy for Covid-19.


Assuntos
Proteína ADAM17 , Enzima de Conversão de Angiotensina 2 , COVID-19 , Colo , Estradiol , Pulmão , Receptores de Estradiol , Proteína ADAM17/genética , Enzima de Conversão de Angiotensina 2/genética , Animais , COVID-19/tratamento farmacológico , COVID-19/virologia , Colo/efeitos dos fármacos , Colo/metabolismo , Estradiol/farmacologia , Feminino , Pulmão/metabolismo , Masculino , Peptidil Dipeptidase A/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Wistar , Receptores de Estradiol/genética , Receptores de Estradiol/metabolismo , SARS-CoV-2/fisiologia , Serina Endopeptidases/genética , Transcrição Genética/efeitos dos fármacos , Internalização do Vírus
16.
Proc Natl Acad Sci U S A ; 119(26): e2122897119, 2022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35700355

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.


Assuntos
Antivirais , Aspartato Carbamoiltransferase , COVID-19 , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante) , Di-Hidro-Orotase , Inibidores Enzimáticos , Pirimidinas , SARS-CoV-2 , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Aspartato Carbamoiltransferase/antagonistas & inibidores , COVID-19/tratamento farmacológico , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/antagonistas & inibidores , Di-Hidro-Orotase/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Inflamação/tratamento farmacológico , Camundongos , Pirimidinas/antagonistas & inibidores , Pirimidinas/biossíntese , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Fator de Transcrição RelA/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
17.
Artigo em Inglês | MEDLINE | ID: mdl-35627870

RESUMO

We aimed to elucidate the range of the incubation period in patients infected with the SARS-CoV-2 Omicron variant in comparison with the Alpha variant. Contact tracing data from three Japanese public health centers (total residents, 1.06 million) collected following the guidelines of the Infectious Diseases Control Law were reviewed for 1589 PCR-confirmed COVID-19 cases diagnosed in January 2022. We identified 77 eligible symptomatic patients for whom the date and setting of transmission were known, in the absence of any other probable routes of transmission. The observed incubation period was 3.03 ± 1.35 days (mean ± SDM). In the log-normal distribution, 5th, 50th and 95th percentile values were 1.3 days (95% CI: 1.0-1.6), 2.8 days (2.5-3.1) and 5.8 days (4.8-7.5), significantly shorter than among the 51 patients with the Alpha variant diagnosed in April and May in 2021 (4.94 days ± 2.19, 2.1 days (1.5-2.7), 4.5 days (4.0-5.1) and 9.6 days (7.4-13.0), p < 0.001). As this incubation period, mainly of sublineage BA.1, is even shorter than that in the Delta variant, it is thought to partially explain the variant replacement occurring in late 2021 to early 2022 in many countries.


Assuntos
COVID-19 , Período de Incubação de Doenças Infecciosas , SARS-CoV-2 , COVID-19/epidemiologia , Busca de Comunicante , Humanos , Japão/epidemiologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia
18.
Viruses ; 14(5)2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35632716

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.


Assuntos
Replicon , SARS-CoV-2 , Antivirais/farmacologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Regiões Promotoras Genéticas , RNA Guia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
19.
Eur J Med Chem ; 238: 114426, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35551037

RESUMO

The COVID-19 pandemic generates a global threat to public health and continuously emerging SARS-CoV-2 variants bring a great challenge to the development of both vaccines and antiviral agents. In this study, we identified UA-18 and its optimized analog UA-30 via the hit-to-lead strategy as novel SARS-CoV-2 fusion inhibitors. The lead compound UA-30 showed potent antiviral activity against infectious SARS-CoV-2 (wuhan-HU-1 variant) in Vero-E6 cells and was also effective against infection of diverse pseudotyped SARS-CoV-2 variants with mutations in the S protein including the Omicron and Delta variants. More importantly, UA-30 might target the cavity between S1 and S2 subunits to stabilize the prefusion state of the SARS-CoV-2 S protein, thus leading to interfering with virus-cell membrane fusion. This study offers a set of novel SARS-CoV-2 fusion inhibitors against SARS-CoV-2 and its variants based on the 3-O-ß-chacotriosyl UA skeleton.


Assuntos
Antivirais , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Triterpenos , Internalização do Vírus , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Triterpenos/farmacologia , Internalização do Vírus/efeitos dos fármacos
20.
J Virol ; 96(11): e0059422, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35543509

RESUMO

It has recently been shown that an early SARS-CoV-2 isolate (NL-02-2020) hijacks interferon-induced transmembrane proteins (IFITMs) for efficient replication in human lung cells, cardiomyocytes, and gut organoids. To date, several "variants of concern" (VOCs) showing increased infectivity and resistance to neutralization have emerged and globally replaced the early viral strains. Here, we determined whether the five current SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron) maintained the dependency on IFITM proteins for efficient replication. We found that depletion of IFITM2 strongly reduces viral RNA production by all VOCs in the human epithelial lung cancer cell line Calu-3. Silencing of IFITM1 had modest effects, while knockdown of IFITM3 resulted in an intermediate phenotype. Strikingly, depletion of IFITM2 generally reduced infectious virus production by more than 4 orders of magnitude. In addition, an antibody directed against the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells, thought to represent major viral target cells in the lung. In conclusion, endogenously expressed IFITM proteins (especially IFITM2) are critical cofactors for efficient replication of genuine SARS-CoV-2 VOCs, including the currently dominant Omicron variant. IMPORTANCE Recent data indicate that SARS-CoV-2 requires endogenously expressed IFITM proteins for efficient infection. However, the results were obtained with an early SARS-CoV-2 isolate. Thus, it remained to be determined whether IFITMs are also important cofactors for infection of emerging SARS-CoV-2 VOCs that outcompeted the original strains in the meantime. This includes the Omicron VOC, which currently dominates the pandemic. Here, we show that depletion of endogenous IFITM2 expression almost entirely prevents productive infection of Alpha, Beta, Gamma, Delta, and Omicron SARS-CoV-2 VOCs in human lung cells. In addition, an antibody targeting the N terminus of IFITM2 inhibited SARS-CoV-2 VOC replication in iPSC-derived alveolar epithelial type II cells. Our results show that SARS-CoV-2 VOCs, including the currently dominant Omicron variant, are strongly dependent on IFITM2 for efficient replication, suggesting a key proviral role of IFITMs in viral transmission and pathogenicity.


Assuntos
Pulmão , Proteínas de Membrana , SARS-CoV-2 , Replicação Viral , COVID-19/virologia , Linhagem Celular Tumoral , Humanos , Pulmão/virologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Internalização do Vírus
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