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2.
STAR Protoc ; 2(4): 100906, 2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34642671

RESUMO

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

3.
Eur J Med Chem ; 226: 113862, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34583312

RESUMO

We report here the synthesis, purification, and characterization of mono- and di-fatty acyl conjugates of remdesivir (RDV) and their in vitro antiviral activity against SAR-CoV-2, an Ebola virus transcription- and replication-competent virus-like particle (trVLP) system, and infectious Ebola virus. The most potent monofatty acyl conjugate was 4b, containing a 4-oxatetradecanolyl at the 3' position. Monofatty acyl conjugates, 3'-O-tetradecanoyl (4a) (IC50(VeroE6) = 2.3 µM; IC50(Calu3) = 0.24 µM), 3'-O-4-oxatetradodecanoyl (4b) (IC50(VeroE6) = 2.0 µM; IC50(Calu3) = 0.18 µM), and 3'-O-(12-ethylthiododecanoyl) (4e) (IC50(VeroE6) = 2.4 µM; IC50(Calu3) = 0.25 µM) derivatives exhibited less activity than RDV (IC50(VeroE6) = 0.85 µM; IC50(Calu3) = 0.06 µM) in both VeroE6 and Calu3 cells. Difatty acylation led to a significant reduction in the antiviral activity of RDV (as shown in conjugates 5a and 5b) against SARS-CoV-2 when compared with monofatty acylation (3a-e and 4a-e). About 77.9% of 4c remained intact after 4 h incubation with human plasma while only 47% of parent RDV was observed at the 2 h time point. The results clearly indicate the effectiveness of fatty acylation to improve the half-life of RDV. The antiviral activities of a number of monofatty acyl conjugates of RDV, such as 3b, 3e, and 4b, were comparable with RDV against the Ebola trVLP system. Meanwhile, the corresponding physical mixtures of RDV and fatty acids 6a and 6b showed 1.6 to 2.2 times less antiviral activity than the corresponding conjugates, 4a and 4c, respectively, against SARS-CoV-2 in VeroE6 cells. A significant reduction in viral RNA synthesis was observed for selected compounds 3a and 4b consistent with the IC50 results. These studies indicate the potential of these compounds as long-acting antiviral agents or prodrugs of RDV.

4.
J Virol ; 95(19): e0065221, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34346762

RESUMO

The filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5' and 3' untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3' UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5' or 3' UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3' UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3' UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3' UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3' UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3' UTR, but not the edited or deletion mutant NP 3' UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3' UTR, VP40 3' UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3' UTR edits also enhanced translation, but neither the wild-type nor the edited 3' UTRs induced IFN. These findings implicate filoviral mRNA 3' UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1. IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3' UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3' UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3' UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.


Assuntos
Regiões 3' não Traduzidas , Adenosina Desaminase/metabolismo , Ebolavirus/genética , Marburgvirus/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Ebolavirus/metabolismo , Genes Reporter , Humanos , Interferon Tipo I/biossíntese , Marburgvirus/metabolismo , MicroRNAs/genética , Mutação , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Polirribossomos/metabolismo , Edição de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
5.
Arch Virol ; 166(12): 3513-3566, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34463877

RESUMO

In March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

6.
Cell Rep ; 36(5): 109479, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34320401

RESUMO

Coronaviruses rely on host membranes for entry, establishment of replication centers, and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we test small molecule inhibitors that target the PI3 kinase VPS34 or fatty acid metabolism for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity. Our studies determine that compounds targeting VPS34 are potent SARS-CoV-2 inhibitors. Mechanistic studies with compounds targeting multiple steps up- and downstream of fatty acid synthase (FASN) identify the importance of triacylglycerol production and protein palmitoylation as requirements for efficient viral RNA synthesis and infectious virus production. Further, FASN knockout results in significantly impaired SARS-CoV-2 replication that can be rescued with fatty acid supplementation. Together, these studies clarify roles for VPS34 and fatty acid metabolism in SARS-CoV-2 replication and identify promising avenues for the development of countermeasures against SARS-CoV-2.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Metabolismo dos Lipídeos/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos , Aminopiridinas/farmacologia , Animais , Células CACO-2 , Linhagem Celular , Chlorocebus aethiops , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ácido Graxo Sintases/efeitos dos fármacos , Ácido Graxo Sintases/genética , Técnicas de Inativação de Genes , Humanos , Lipoilação/efeitos dos fármacos , Pirimidinas/farmacologia , RNA Viral/metabolismo , Triglicerídeos/metabolismo , Células Vero
7.
EMBO J ; 40(18): e105658, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34260076

RESUMO

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.

8.
iScience ; 24(6): 102681, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34095780

RESUMO

Nucleocapsid (N) encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays key roles in the replication cycle and is a critical serological marker. Here, we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high-affinity RNA-binding platform. We also map the RNA-binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose coils, showing how N-RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based on antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.

9.
Antiviral Res ; 191: 105088, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34019950

RESUMO

3-deazaneplanocin A (DzNep) and its 3-brominated analogs inhibit replication of several RNA viruses. This antiviral activity is attributed to inhibition of S-adenosyl homocysteine hydrolase (SAHase) and consequently inhibition of viral methyltransferases, impairing translation of viral transcripts. The L-enantiomers of some derivatives retain antiviral activity despite dramatically reduced inhibition of SAHase in vitro. To better understand the mechanisms by which these compounds exert their antiviral effects, we compared DzNep, its 3-bromo-derivative, CL123, and the related enantiomers, CL4033 and CL4053, for their activities towards the model negative-sense RNA virus vesicular stomatitis virus (VSV). In cell culture, DzNep, CL123 and CL4033 each exhibited 50 percent inhibitory concentrations (IC50s) in the nanomolar range whereas the IC50 for the L-form, CL4053, was 34-85 times higher. When a CL123-resistant mutant (VSVR) was selected, it exhibited cross-resistance to each of the neplanocin analogs, but retained sensitivity to the adenosine analog BCX4430, an RNA chain terminator. Sequencing of VSVR identified a mutation in the C-terminal domain (CTD) of the viral large (L) protein, a domain implicated in regulation of L protein methyltransferase activity. CL123 inhibited VSV viral mRNA 5' cap methylation, impaired viral protein synthesis and decreased association of viral mRNAs with polysomes. Modest impacts on viral transcription were also demonstrated. VSVR exhibited partial resistance in each of these assays but its replication was impaired, relative to the parent VSV, in the absence of the inhibitors. These data suggest that DzNep, CL123 and CL4033 inhibit VSV through impairment of viral mRNA cap methylation and that the L-form, CL4053, based on the cross-resistance of VSVR, may act by a similar mechanism.

10.
Viruses ; 13(3)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799842

RESUMO

The ongoing SARS-CoV-2 pandemic has resulted in an increased need for technologies capable of efficiently disinfecting public spaces as well as personal protective equipment. UV light disinfection is a well-established method for inactivating respiratory viruses. Here, we have determined that broad-spectrum, pulsed UV light is effective at inactivating SARS-CoV-2 on multiple surfaces in vitro. For hard, non-porous surfaces, we observed that SARS-CoV-2 was inactivated to undetectable levels on plastic and glass with a UV dose of 34.9 mJ/cm2 and stainless steel with a dose of 52.5 mJ/cm2. We also observed that broad-spectrum, pulsed UV light is effective at reducing SARS-CoV-2 on N95 respirator material to undetectable levels with a dose of 103 mJ/cm2. We included UV dosimeter cards that provide a colorimetric readout of UV dose and demonstrated their utility as a means to confirm desired levels of exposure were reached. Together, the results presented here demonstrate that broad-spectrum, pulsed UV light is an effective technology for the in vitro inactivation of SARS-CoV-2 on multiple surfaces.


Assuntos
COVID-19/virologia , Desinfecção/métodos , Máscaras/virologia , SARS-CoV-2/efeitos da radiação , Inativação de Vírus/efeitos da radiação , COVID-19/prevenção & controle , Desinfecção/instrumentação , Humanos , SARS-CoV-2/fisiologia , Raios Ultravioleta
11.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649232

RESUMO

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNß messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.


Assuntos
Proteína DEAD-box 58 , Helicase IFIH1 Induzida por Interferon , Interferon beta , Receptores Imunológicos , Proteínas não Estruturais Virais , Cristalografia por Raios X , Proteína DEAD-box 58/química , Proteína DEAD-box 58/metabolismo , Medição da Troca de Deutério , Células HEK293 , Humanos , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/química , Interferon beta/metabolismo , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
12.
Nat Commun ; 12(1): 28, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397924

RESUMO

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.


Assuntos
Núcleo Celular/metabolismo , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Drosophila/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Células-Tronco Neurais/metabolismo , Sinais de Localização Nuclear/metabolismo , Mutação Puntual/genética , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Fatores de Transcrição SOXB1/genética , Relação Estrutura-Atividade
13.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33408171

RESUMO

Infection with Zaire ebolavirus (EBOV), a member of the Filoviridae family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 105 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 104 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge.IMPORTANCE Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV.


Assuntos
Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/imunologia , Tecido Linfoide/imunologia , Proteínas Virais Reguladoras e Acessórias/imunologia , Imunidade Adaptativa , Animais , Antivirais/sangue , Ebolavirus/genética , Ebolavirus/imunologia , Regulação da Expressão Gênica/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Tecido Linfoide/virologia , Macaca fascicularis , Mutação , Baço/imunologia , Transcriptoma , Proteínas Virais Reguladoras e Acessórias/genética
14.
mSphere ; 5(6)2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33328346

RESUMO

The Nipah virus (NiV) phosphoprotein (P) gene encodes four proteins. Three of these-P, V, and W-possess a common N-terminal domain but distinct C termini. These proteins interact with immune modulators. Previous studies demonstrated that P, V, and W bind STAT1 and STAT4 and that V also interacts with STAT2 but not with STAT3. The STAT1 and STAT2 interactions block interferon (IFN)-induced STAT tyrosine phosphorylation. To more fully characterize the interactions of P, V, and W with the STATs, we screened for interaction of each viral protein with STATs 1 to 6 by coimmunoprecipitation. We demonstrate that NiV P, V, and W interact with STAT4 through their common N-terminal domain and block STAT4 activity, based on a STAT4 response element reporter assay. Although none of the NiV proteins interact with STAT3 or STAT6, NiV V, but not P or W, interacts with STAT5 through its unique C terminus. Furthermore, the interaction of NiV V with STAT5 was not disrupted by overexpression of the N-terminal binding STAT1 or the C-terminal binding MDA5. NiV V also inhibits a STAT5 response element reporter assay. Residues 114 to 140 of the common N-terminal domain of the NiV P gene products were found to be sufficient to bind STAT1 and STAT4. Analysis of STAT1-STAT3 chimeras suggests that the P gene products target the STAT1 SH2 domain. When fused to GST, the 114-140 peptide is sufficient to decrease STAT1 phosphorylation in IFN-ß-stimulated cells, suggesting that this peptide could potentially be fused to heterologous proteins to confer inhibition of STAT1- and STAT4-dependent responses.IMPORTANCE How Nipah virus (NiV) antagonizes innate immune responses is incompletely understood. The P gene of NiV encodes the P, V, and W proteins. These proteins have a common N-terminal sequence that is sufficient to bind to STAT1 and STAT2 and block IFN-induced signal transduction. This study sought to more fully understand how P, V, and W engage with the STAT family of transcription factors to influence their functions. The results identify a novel interaction of V with STAT5 and demonstrate V inhibition of STAT5 function. We also demonstrate that the common N-terminal residues 114 to 140 of P, V, and W are critical for inhibition of STAT1 and STAT4 function, map the interaction to the SH2 region of STAT1, and show that a fusion construct with this peptide significantly inhibits cytokine-induced STAT1 phosphorylation. These data clarify how these important virulence factors modulate innate antiviral defenses.


Assuntos
Núcleo Celular/química , Infecções por Henipavirus/metabolismo , Vírus Nipah/fisiologia , Fatores de Transcrição STAT/metabolismo , Proteínas Virais/metabolismo , Células HEK293 , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Humanos , Imunidade Inata/imunologia , Fosforilação , Fatores de Transcrição STAT/genética , Transdução de Sinais , Transativadores/metabolismo , Proteínas Virais/genética
15.
bioRxiv ; 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33269347

RESUMO

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

16.
ACS Infect Dis ; 6(10): 2783-2799, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32870648

RESUMO

Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have been approved for MARV disease. The MARV matrix protein VP40 (mVP40) plays central roles in virus assembly and budding. mVP40 also inhibits interferon signaling by inhibiting the function of Janus kinase 1. This suppression of host antiviral defenses likely contributes to MARV virulence and therefore is a potential therapeutic target. We developed and optimized a cell-based high-throughput screening (HTS) assay in 384-well format to measure mVP40 interferon (IFN) antagonist function such that inhibitors could be identified. We performed a pilot screen of 1280 bioactive compounds and identified 3 hits, azaguanine-8, tosufloxacin hydrochloride, and linezolid, with Z scores > 3 and no significant cytotoxicity. Of these, azaguanine-8 inhibited MARV growth at noncytotoxic concentrations. These data demonstrate the suitability of the HTS mVP40 assay for drug discovery and suggest potential directions for anti-MARV therapeutic development.


Assuntos
Doença do Vírus de Marburg , Marburgvirus , Animais , Ensaios de Triagem em Larga Escala , Humanos , Interferons , Montagem de Vírus
17.
bioRxiv ; 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32743584

RESUMO

Therapeutics targeting replication of SARS coronavirus 2 (SARS-CoV-2) are urgently needed. Coronaviruses rely on host membranes for entry, establishment of replication centers and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we tested small molecule inhibitors that target membrane dynamics or lipid metabolism. Included were inhibitors of the PI3 kinase VPS34, which functions in autophagy, endocytosis and other processes; Orlistat, an inhibitor of lipases and fatty acid synthetase, is approved by the FDA as a treatment for obesity; and Triacsin C which inhibits long chain fatty acyl-CoA synthetases. VPS34 inhibitors, Orlistat and Triacsin C inhibited virus growth in Vero E6 cells and in the human airway epithelial cell line Calu-3, acting at a post-entry step in the virus replication cycle. Of these the VPS34 inhibitors exhibit the most potent activity.

18.
Viruses ; 12(6)2020 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-32517266

RESUMO

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Ensaio de Placa Viral/métodos , Inativação de Vírus , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/patogenicidade , COVID-19 , Celulose , Chlorocebus aethiops , Meios de Cultura/química , Formaldeído , Guanidinas/farmacologia , Células HEK293 , Humanos , Pandemias , Fenóis/farmacologia , Propiolactona/farmacologia , SARS-CoV-2 , Sefarose , Células Vero
19.
J Virol ; 94(13)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32295912

RESUMO

Menglà virus (MLAV), identified in Rousettus bats, is a phylogenetically distinct member of the family Filoviridae Because the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) modulate host innate immunity, MLAV VP35, VP40, and VP24 proteins were compared with their EBOV and MARV homologs for innate immune pathway modulation. In human and Rousettus cells, MLAV VP35 behaved like EBOV and MARV VP35s, inhibiting virus-induced activation of the interferon beta (IFN-ß) promoter and interferon regulatory factor 3 (IRF3) phosphorylation. MLAV VP35 also interacted with PACT, a host protein engaged by EBOV VP35 to inhibit RIG-I signaling. MLAV VP35 also inhibits PKR activation. MLAV VP40 was demonstrated to inhibit type I IFN-induced gene expression in human and bat cells. It blocked STAT1 tyrosine phosphorylation induced either by type I IFN or overexpressed Jak1, paralleling MARV VP40. MLAV VP40 also inhibited virus-induced IFN-ß promoter activation, a property shared by MARV VP40 and EBOV VP24. A Jak kinase inhibitor did not recapitulate this inhibition in the absence of viral proteins. Therefore, inhibition of Jak-STAT signaling is insufficient to explain inhibition of IFN-ß promoter activation. MLAV VP24 did not inhibit IFN-induced gene expression or bind karyopherin α proteins, properties of EBOV VP24. MLAV VP24 differed from MARV VP24 in that it failed to interact with Keap1 or activate an antioxidant response element reporter gene due to the absence of a Keap1-binding motif. These functional observations support a closer relationship of MLAV to MARV than to EBOV but also are consistent with MLAV belonging to a distinct genus.IMPORTANCE EBOV and MARV, members of the family Filoviridae, are highly pathogenic zoonotic viruses that cause severe disease in humans. Both viruses use several mechanisms to modulate the host innate immune response, and these likely contribute to the severity of disease. Here, we demonstrate that MLAV, a filovirus newly discovered in a bat, suppresses antiviral type I interferon responses in both human and bat cells. Inhibitory activities are possessed by MLAV VP35 and VP40, which parallels how MARV blocks IFN responses. However, whereas MARV activates cellular antioxidant responses through an interaction between its VP24 protein and host protein Keap1, MLAV VP24 lacks a Keap1-binding motif and fails to activate this cytoprotective response. These data indicate that MLAV possesses immune-suppressing functions that could facilitate human infection. They also support the placement of MLAV in a different genus than either EBOV or MARV.


Assuntos
Infecções por Filoviridae/fisiopatologia , Filoviridae/genética , Animais , Quirópteros/imunologia , Quirópteros/virologia , Ebolavirus , Filoviridae/metabolismo , Filoviridae/patogenicidade , Células HEK293 , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Marburgvirus , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Transcrição STAT1 , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
20.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32321809

RESUMO

Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus in the Paramyxoviridae family, are recently emerged, highly lethal zoonotic pathogens. The NiV and HeV nonsegmented, negative-sense RNA genomes encode nine proteins, including the W protein. Expressed from the P gene through mRNA editing, W shares a common N-terminus with P and V but has a unique C-terminus. Expressed alone, W modulates innate immune responses by several mechanisms, and elimination of W from NiV alters the course of infection in experimentally infected ferrets. However, the specific host interactions that allow W to modulate innate immunity are incompletely understood. This study demonstrates that the NiV and HeV W proteins interact with all seven isoforms of the 14-3-3 family, regulatory molecules that preferentially bind phosphorylated target proteins to regulate a wide range of cellular functions. The interaction is dependent on the penultimate amino acid residue in the W sequence, a conserved, phosphorylated serine. The cocrystal structure of the W C-terminal binding motif with 14-3-3 provides only the second structure of a complex containing a mode III interactor, which is defined as a 14-3-3 interaction with a phosphoserine/phosphothreonine at the C-termini of the target protein. Transcriptomic analysis of inducible cell lines infected with an RNA virus and expressing either wild-type W or W lacking 14-3-3 binding, identifies new functions for W. These include the regulation of cellular metabolic processes, extracellular matrix organization, and apoptosis.IMPORTANCE Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus, are recently emerged, highly lethal zoonotic pathogens that cause yearly outbreaks. NiV and HeV each encode a W protein that has roles in regulating host signaling pathways, including antagonism of the innate immune response. However, the mechanisms used by W to regulate these host responses are not clear. Here, characterization of the interaction of NiV and HeV W with 14-3-3 identifies modulation of 14-3-3-regulated host signaling pathways not previously associated with W, suggesting new avenues of research. The cocrystal structure of the NiV W:14-3-3 complex, as only the second structure of a 14-3-3 mode III interactor, provides further insight into this less-well-understood 14-3-3 binding motif.


Assuntos
Proteínas 14-3-3/metabolismo , Regulação da Expressão Gênica , Vírus Hendra/metabolismo , Infecções por Henipavirus/metabolismo , Vírus Nipah/metabolismo , Proteínas Virais/metabolismo , Proteínas 14-3-3/genética , Células HEK293 , Vírus Hendra/genética , Infecções por Henipavirus/genética , Humanos , Vírus Nipah/genética , Proteínas Virais/genética
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