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1.
Proc Natl Acad Sci U S A ; 118(36)2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34479996

RESUMO

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

2.
EMBO Rep ; 22(10): e52823, 2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34397140

RESUMO

Interferon (IFN) induction of IFN-stimulated genes (ISGs) creates a formidable protective antiviral state. However, loss of appropriate control mechanisms can result in constitutive pathogenic ISG upregulation. Here, we used genome-scale loss-of-function screening to establish genes critical for IFN-induced transcription, identifying all expected members of the JAK-STAT signaling pathway and a previously unappreciated epigenetic reader, bromodomain-containing protein 9 (BRD9), the defining subunit of non-canonical BAF (ncBAF) chromatin-remodeling complexes. Genetic knockout or small-molecule-mediated degradation of BRD9 limits IFN-induced expression of a subset of ISGs in multiple cell types and prevents IFN from exerting full antiviral activity against several RNA and DNA viruses, including influenza virus, human immunodeficiency virus (HIV1), and herpes simplex virus (HSV1). Mechanistically, BRD9 acts at the level of transcription, and its IFN-triggered proximal association with the ISG transcriptional activator, STAT2, suggests a functional localization at selected ISG promoters. Furthermore, BRD9 relies on its intact acetyl-binding bromodomain and unique ncBAF scaffolding interaction with GLTSCR1/1L to promote IFN action. Given its druggability, BRD9 is an attractive target for dampening ISG expression under certain autoinflammatory conditions.


Assuntos
Antivirais , Interferons , Antivirais/farmacologia , Expressão Gênica , Humanos , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Fatores de Transcrição/genética
3.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34319869

RESUMO

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , COVID-19/tratamento farmacológico , COVID-19/virologia , Quimases/antagonistas & inibidores , Compostos de Fenilureia/farmacologia , Quinolinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Animais , Antivirais/farmacologia , COVID-19/enzimologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade
4.
PLoS Pathog ; 17(6): e1009644, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138976

RESUMO

Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Vírus da Hepatite Murina/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Fator 6 Ativador da Transcrição/metabolismo , Animais , Antivirais/uso terapêutico , Linhagem Celular , Chlorocebus aethiops , Sistemas de Liberação de Medicamentos , Endorribonucleases/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , RNA-Seq , Células Vero , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
5.
Nat Nanotechnol ; 16(8): 918-925, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34083772

RESUMO

Minimizing the spread of viruses in the environment is the first defence line when fighting outbreaks and pandemics, but the current COVID-19 pandemic demonstrates how difficult this is on a global scale, particularly in a sustainable and environmentally friendly way. Here we introduce and develop a sustainable and biodegradable antiviral filtration membrane composed of amyloid nanofibrils made from food-grade milk proteins and iron oxyhydroxide nanoparticles synthesized in situ from iron salts by simple pH tuning. Thus, all the membrane components are made of environmentally friendly, non-toxic and widely available materials. The membrane has outstanding efficacy against a broad range of viruses, which include enveloped, non-enveloped, airborne and waterborne viruses, such as SARS-CoV-2, H1N1 (the influenza A virus strain responsible for the swine flu pandemic in 2009) and enterovirus 71 (a non-enveloped virus resistant to harsh conditions, such as highly acidic pH), which highlights a possible role in fighting the current and future viral outbreaks and pandemics.


Assuntos
Amiloide/química , Antivirais/farmacologia , Compostos Férricos/química , Filtros Microporos , Nanopartículas/química , Amiloide/farmacologia , Antivirais/química , Compostos Férricos/farmacologia , Humanos , Lactoglobulinas/química , Filtros Microporos/virologia , Inativação de Vírus/efeitos dos fármacos , Vírus/classificação , Vírus/efeitos dos fármacos , Vírus/isolamento & purificação , Purificação da Água
6.
Elife ; 102021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34003114

RESUMO

When culturing SARS-CoV-2 in the laboratory it is vital to avoid deletions in the gene for the spike protein that could affect the interpretation of experiments.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Chlorocebus aethiops , Humanos , SARS-CoV-2
7.
J Exp Med ; 218(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33882122

RESUMO

The disease severity of influenza is highly variable in humans, and one genetic determinant behind these differences is the IFITM3 gene. As an effector of the interferon response, IFITM3 potently blocks cytosolic entry of influenza A virus (IAV). Here, we reveal a novel level of inhibition by IFITM3 in vivo: We show that incorporation of IFITM3 into IAV particles competes with incorporation of viral hemagglutinin (HA). Decreased virion HA levels did not reduce infectivity, suggesting that high HA density on IAV virions may be an antagonistic strategy used by the virus to prevent direct inhibition. However, we found that IFITM3-mediated reduction in HA content sensitizes IAV to antibody-mediated neutralization. Mathematical modeling predicted that this effect decreases and delays peak IAV titers, and we show that, indeed, IFITM3-mediated sensitization of IAV to antibody-mediated neutralization impacts infection outcome in an in vivo mouse model. Overall, our data describe a previously unappreciated interplay between the innate effector IFITM3 and the adaptive immune response.


Assuntos
Anticorpos Neutralizantes/imunologia , Vírus da Influenza A/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Células A549 , Imunidade Adaptativa/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cães , Feminino , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Influenza Humana/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteólise
8.
Viruses ; 13(3)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806893

RESUMO

SUMOylation is a highly dynamic ubiquitin-like post-translational modification that is essential for cells to respond to and resolve various genotoxic and proteotoxic stresses. Virus infections also constitute a considerable stress scenario for cells, and recent research has started to uncover the diverse roles of SUMOylation in regulating virus replication, not least by impacting antiviral defenses. Here, we review some of the key findings of this virus-host interplay, and discuss the increasingly important contribution that large-scale, unbiased, proteomic methodologies are making to discoveries in this field. We highlight the latest proteomic technologies that have been specifically developed to understand SUMOylation dynamics in response to cellular stresses, and comment on how these techniques might be best applied to dissect the biology of SUMOylation during innate immunity. Furthermore, we showcase a selection of studies that have already used SUMO proteomics to reveal novel aspects of host innate defense against viruses, such as functional cross-talk between SUMO proteins and other ubiquitin-like modifiers, viral antagonism of SUMO-modified antiviral restriction factors, and an infection-triggered SUMO-switch that releases endogenous retroelement RNAs to stimulate antiviral interferon responses. Future research in this area has the potential to provide new and diverse mechanistic insights into host immune defenses.


Assuntos
Interações entre Hospedeiro e Microrganismos , Imunidade Inata , Sumoilação , Viroses , Humanos , Proteômica , Viroses/imunologia , Viroses/virologia , Replicação Viral
9.
Trends Microbiol ; 29(11): 973-982, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33757684

RESUMO

Pandemics are caused by novel pathogens to which pre-existing antibody immunity is lacking. Under these circumstances, the body must rely on innate interferon-mediated defenses to limit pathogen replication and allow development of critical humoral protection. Here, we highlight studies on disease susceptibility during H1N1 influenza and COVID-19 (SARS-CoV-2) pandemics. An emerging concept is that genetic and non-genetic deficiencies in interferon system components lead to uncontrolled virus replication and severe illness in a subset of people. Intriguingly, new findings suggest that individuals with autoantibodies neutralizing the antiviral function of interferon are at increased risk of severe COVID-19. We discuss key questions surrounding how such autoantibodies develop and function, as well as the general implications of diagnosing interferon deficiencies for personalized therapies.


Assuntos
Resistência à Doença , Interações Hospedeiro-Patógeno , Interferons/metabolismo , Viroses/etiologia , Viroses/metabolismo , Alelos , Animais , Anticorpos Neutralizantes/imunologia , Autoanticorpos/imunologia , Autoimunidade , Progressão da Doença , Resistência à Doença/imunologia , Suscetibilidade a Doenças , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferons/antagonistas & inibidores , Interferons/imunologia , Mutação com Perda de Função , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de Doença , Viroses/diagnóstico , Viroses/epidemiologia
10.
PLoS Biol ; 19(3): e3001006, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33760807

RESUMO

Since entering the human population, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; the causative agent of Coronavirus Disease 2019 [COVID-19]) has spread worldwide, causing >100 million infections and >2 million deaths. While large-scale sequencing efforts have identified numerous genetic variants in SARS-CoV-2 during its circulation, it remains largely unclear whether many of these changes impact adaptation, replication, or transmission of the virus. Here, we characterized 14 different low-passage replication-competent human SARS-CoV-2 isolates representing all major European clades observed during the first pandemic wave in early 2020. By integrating viral sequencing data from patient material, virus stocks, and passaging experiments, together with kinetic virus replication data from nonhuman Vero-CCL81 cells and primary differentiated human bronchial epithelial cells (BEpCs), we observed several SARS-CoV-2 features that associate with distinct phenotypes. Notably, naturally occurring variants in Orf3a (Q57H) and nsp2 (T85I) were associated with poor replication in Vero-CCL81 cells but not in BEpCs, while SARS-CoV-2 isolates expressing the Spike D614G variant generally exhibited enhanced replication abilities in BEpCs. Strikingly, low-passage Vero-derived stock preparation of 3 SARS-CoV-2 isolates selected for substitutions at positions 5/6 of E and were highly attenuated in BEpCs, revealing a key cell-specific function to this region. Rare isolate-specific deletions were also observed in the Spike furin cleavage site during Vero-CCL81 passage, but these were rapidly selected against in BEpCs, underscoring the importance of this site for SARS-CoV-2 replication in primary human cells. Overall, our study uncovers sequence features in SARS-CoV-2 variants that determine cell-specific replication and highlights the need to monitor SARS-CoV-2 stocks carefully when phenotyping newly emerging variants or potential variants of concern.


Assuntos
SARS-CoV-2/fisiologia , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Sequência de Bases , Brônquios/patologia , COVID-19/diagnóstico , COVID-19/virologia , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/patologia , Células Epiteliais/virologia , Furina/metabolismo , Interações Hospedeiro-Patógeno , Humanos , SARS-CoV-2/isolamento & purificação , Células Vero
11.
mBio ; 11(5)2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32913009

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), is a recently emerged respiratory coronavirus that has infected >23 million people worldwide with >800,000 deaths. Few COVID-19 therapeutics are available, and the basis for severe infections is poorly understood. Here, we investigated properties of type I (ß), II (γ), and III (λ1) interferons (IFNs), potent immune cytokines that are normally produced during infection and that upregulate IFN-stimulated gene (ISG) effectors to limit virus replication. IFNs are already in clinical trials to treat COVID-19. However, recent studies highlight the potential for IFNs to enhance expression of host angiotensin-converting enzyme 2 (ACE2), suggesting that IFN therapy or natural coinfections could exacerbate COVID-19 by upregulating this critical virus entry receptor. Using a cell line model, we found that beta interferon (IFN-ß) strongly upregulated expression of canonical antiviral ISGs, as well as ACE2 at the mRNA and cell surface protein levels. Strikingly, IFN-λ1 upregulated antiviral ISGs, but ACE2 mRNA was only marginally elevated and did not lead to detectably increased ACE2 protein at the cell surface. IFN-γ induced the weakest ISG response but clearly enhanced surface expression of ACE2. Importantly, all IFN types inhibited SARS-CoV-2 replication in a dose-dependent manner, and IFN-ß and IFN-λ1 exhibited potent antiviral activity in primary human bronchial epithelial cells. Our data imply that type-specific mechanisms or kinetics shape IFN-enhanced ACE2 transcript and cell surface levels but that the antiviral action of IFNs against SARS-CoV-2 counterbalances any proviral effects of ACE2 induction. These insights should aid in evaluating the benefits of specific IFNs, particularly IFN-λ, as repurposed therapeutics.IMPORTANCE Repurposing existing, clinically approved, antiviral drugs as COVID-19 therapeutics is a rapid way to help combat the SARS-CoV-2 pandemic. Interferons (IFNs) usually form part of the body's natural innate immune defenses against viruses, and they have been used with partial success to treat previous new viral threats, such as HIV, hepatitis C virus, and Ebola virus. Nevertheless, IFNs can have undesirable side effects, and recent reports indicate that IFNs upregulate the expression of host ACE2 (a critical entry receptor for SARS-CoV-2), raising the possibility that IFN treatments could exacerbate COVID-19. Here, we studied the antiviral- and ACE2-inducing properties of different IFN types in both a human lung cell line model and primary human bronchial epithelial cells. We observed differences between IFNs with respect to their induction of antiviral genes and abilities to enhance the cell surface expression of ACE2. Nevertheless, all the IFNs limited SARS-CoV-2 replication, suggesting that their antiviral actions can counterbalance increased ACE2.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Interferons/farmacologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Idoso , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus/imunologia , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Imunoterapia/métodos , Interferon Tipo I/efeitos adversos , Interferon gama/efeitos adversos , Interferons/efeitos adversos , Pandemias , Peptidil Dipeptidase A/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Virais/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , SARS-CoV-2 , Regulação para Cima/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
12.
mSphere ; 5(4)2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848003

RESUMO

Natural adaptation of an antigenically novel avian influenza A virus (IAV) to be transmitted efficiently in humans has the potential to trigger a devastating pandemic. Understanding viral genetic determinants underlying adaptation is therefore critical for pandemic preparedness, as the knowledge gained enhances surveillance and eradication efforts, prepandemic vaccine design, and efficacy assessment of antivirals. However, this work has risks, as making gain-of-function substitutions in fully infectious IAVs may create a pathogen with pandemic potential. Thus, such experiments must be tightly controlled through physical and biological risk mitigation strategies. Here, we applied a previously described biological containment system for IAVs to a 2009 pandemic H1N1 strain and a highly pathogenic H5N1 strain. The system relies on deletion of the essential viral hemagglutinin (HA) gene, which is instead provided in trans, thereby restricting multicycle virus replication to genetically modified HA-complementing cells. In place of HA, a Renilla luciferase gene is inserted within the viral genome, and a live-cell luciferase substrate allows real-time quantitative monitoring of viral replication kinetics with a high dynamic range. We demonstrate that biologically contained IAV-like particles exhibit wild-type sensitivities to approved antivirals, including oseltamivir, zanamivir, and baloxavir. Furthermore, the inability of these IAV-like particles to genetically acquire the host-encoded HA allowed us to introduce gain-of-function substitutions in the H5 HA gene that promote mammalian transmissibility. Biologically contained "transmissible" H5N1 IAV-like particles exhibited wild-type sensitivities to approved antivirals, to the fusion inhibitor S20, and to neutralization by existing H5 monoclonal and polyclonal sera. This work represents a proof of principle that biologically contained IAV systems can be used to safely conduct selected gain-of-function experiments.IMPORTANCE Understanding how animal influenza viruses can adapt to spread in humans is critical to prepare for, and prevent, new pandemics. However, working safely with pathogens that have pandemic potential requires tight regulation and the use of high-level physical and biological risk mitigation strategies to stop accidental loss of containment. Here, we used a biological containment system for influenza viruses to study strains with pandemic potential. The system relies on deletion of the essential HA gene from the viral genome and its provision by a genetically modified cell line, to which virus propagation is therefore restricted. We show that this method permits safe handling of these pathogens, including gain-of-function variants, without the risk of generating fully infectious viruses. Furthermore, we demonstrate that this system can be used to assess virus sensitivity to both approved and experimental drugs, as well as the antigenic profile of viruses, important considerations for evaluating prepandemic vaccine and antiviral strategies.


Assuntos
Adaptação Fisiológica/genética , Mutação com Ganho de Função , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Pandemias/prevenção & controle , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Deleção de Genes , Genes Reporter , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Estudo de Prova de Conceito , Replicação Viral
13.
Proc Natl Acad Sci U S A ; 116(35): 17399-17408, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31391303

RESUMO

Dynamic small ubiquitin-like modifier (SUMO) linkages to diverse cellular protein groups are critical to orchestrate resolution of stresses such as genome damage, hypoxia, or proteotoxicity. Defense against pathogen insult (often reliant upon host recognition of "non-self" nucleic acids) is also modulated by SUMO, but the underlying mechanisms are incompletely understood. Here, we used quantitative SILAC-based proteomics to survey pan-viral host SUMOylation responses, creating a resource of almost 600 common and unique SUMO remodeling events that are mounted during influenza A and B virus infections, as well as during viral innate immune stimulation. Subsequent mechanistic profiling focused on a common infection-induced loss of the SUMO-modified form of TRIM28/KAP1, a host transcriptional repressor. By integrating knockout and reconstitution models with system-wide transcriptomics, we provide evidence that influenza virus-triggered loss of SUMO-modified TRIM28 leads to derepression of endogenous retroviral (ERV) elements, unmasking this cellular source of "self" double-stranded (ds)RNA. Consequently, loss of SUMO-modified TRIM28 potentiates canonical cytosolic dsRNA-activated IFN-mediated defenses that rely on RIG-I, MAVS, TBK1, and JAK1. Intriguingly, although wild-type influenza A virus robustly triggers this SUMO switch in TRIM28, the induction of IFN-stimulated genes is limited unless expression of the viral dsRNA-binding protein NS1 is abrogated. This may imply a viral strategy to antagonize such a host response by sequestration of induced immunostimulatory ERV dsRNAs. Overall, our data reveal that a key nuclear mechanism that normally prevents aberrant expression of ERV elements (ERVs) has been functionally co-opted via a stress-induced SUMO switch to augment antiviral immunity.


Assuntos
Retrovirus Endógenos/imunologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Interações Microbianas , Proteína SUMO-1/metabolismo , Animais , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/imunologia , Modelos Biológicos , RNA de Cadeia Dupla/metabolismo , Sumoilação , Proteína 28 com Motivo Tripartido/metabolismo , Replicação Viral
14.
Nat Commun ; 10(1): 3396, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363119

RESUMO

Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPol-promoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.


Assuntos
Vírus da Influenza A/enzimologia , Influenza Aviária/genética , Splicing de RNA , Proteínas de Ligação a RNA/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Aves , Galinhas , Humanos , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Influenza Aviária/metabolismo , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Proteínas Nucleares , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral
15.
Nature ; 567(7746): 109-112, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787439

RESUMO

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.


Assuntos
Quirópteros/virologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Especificidade de Hospedeiro , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Zoonoses/imunologia , Zoonoses/virologia , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Galinhas/genética , Galinhas/imunologia , Quirópteros/genética , Quirópteros/imunologia , Quirópteros/metabolismo , Feminino , Perfilação da Expressão Gênica , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Especificidade de Hospedeiro/genética , Especificidade de Hospedeiro/imunologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Sistema Respiratório/virologia , Suínos/genética , Suínos/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Replicação Viral , Zoonoses/genética , Zoonoses/metabolismo
16.
J Virol ; 92(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29237829

RESUMO

Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85ß, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85ß interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85ß-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs.IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.


Assuntos
Quirópteros/virologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Influenza A/metabolismo , Proteínas não Estruturais Virais/fisiologia , Células A549 , Substituição de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Quirópteros/genética , Surtos de Doenças , Cães , Células HEK293 , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Redes e Vias Metabólicas/genética , Modelos Moleculares , Infecções por Orthomyxoviridae/virologia , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA de Cadeia Dupla , Transdução de Sinais , Especificidade da Espécie , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genética
17.
Cell Rep ; 20(11): 2538-2546, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-28903035

RESUMO

Host restriction of influenza A virus limits pandemic emergence. The viral RNA polymerase (vPol) is an essential enzyme that must adapt for avian viruses to replicate in humans. Species differences in host ANP32A dictate adaptation: human ANP32A lacks an uncharacterized 33 amino-acid insertion that is present in avian ANP32A. Here, we uncover important contributions of host SUMOylation to vPol activity, including avANP32A function. We also identify a hydrophobic SUMO interaction motif (SIM)-like sequence unique to avANP32A that critically supports avian-signature vPol. Unrelated SIM sequences partially recapitulate this function when introduced into huANP32A. By investigating ANP32A-vPol interactions, we find that huANP32A interacts weakly with both human- and avian-signature vPols, while the hydrophobic motif of avANP32A promotes stronger interactions. Furthermore, we identify a highly acidic stretch in avANP32A that constitutes a major site of vPol interaction. Our data suggest compensatory mechanisms underlying vPol adaptation to host ANP32A independent of species-specific interactions.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas Nucleares , Proteínas de Ligação a RNA , Sumoilação
18.
J Gen Virol ; 98(9): 2267-2273, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28869005

RESUMO

NS1 proteins of influenza A and B viruses share limited sequence homology, yet both are potent manipulators of host cell processes, particularly interferon (IFN) induction. Although many cellular partners are reported for A/NS1, only a few (e.g. PKR and ISG15) have been identified for B/NS1. Here, affinity-purification and mass spectrometry were used to expand the known host interactome of B/NS1. We identified 22 human proteins as new putative targets for B/NS1, validating several, including DHX9, ILF3, YBX1 and HNRNPC. Consistent with two RNA-binding domains in B/NS1, many of the identified factors bind RNA and some interact with B/NS1 in an RNA-dependent manner. Functional characterization of several B/NS1 interactors identified SNRNP200 as a potential positive regulator of host IFN responses, while ILF3 exhibited dual roles in both IFN induction and influenza B virus replication. These data provide a resource for future investigations into the mechanisms underpinning host cell modulation by influenza B virus NS1.


Assuntos
Vírus da Influenza B/isolamento & purificação , Influenza Humana/metabolismo , Proteínas não Estruturais Virais/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Humanos , Vírus da Influenza B/genética , Vírus da Influenza B/metabolismo , Influenza Humana/genética , Influenza Humana/virologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Fator Nuclear 90/genética , Proteínas do Fator Nuclear 90/metabolismo , Ligação Proteica , Proteínas não Estruturais Virais/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo
19.
J Virol ; 91(21)2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28814525

RESUMO

Rational characterization of virulence and host-adaptive markers in the multifunctional influenza A virus NS1 protein is hindered by a lack of comprehensive knowledge about NS1-host protein protein interfaces. Here, we surveyed the impact of amino acid variation in NS1 at its structurally defined binding site for host p85ß, a regulator of phosphoinositide 3-kinase (PI3K) signaling. Structure-guided alanine scanning of all viral residues at this interface defined 10 positions contributing to the interaction, with residues 89, 95, 98, 133, 145, and 162 being the most important. A bioinformatic study of >24,000 publicly available NS1 sequences derived from viruses infecting different hosts highlighted several prevalent amino acid variants at the p85ß interface that either enhanced (I95) or weakened (N135, T145, L161, Y161, S164) p85ß binding. Interestingly, analysis of viruses circulating in humans since the 1918 pandemic revealed the temporal acquisition of functionally relevant variants at this interface. I95 (which enhanced p85ß binding) quickly became prevalent in the 1940s and experimentally conferred a fitness advantage to a recombinant 1930s-based H1N1 virus in human lung epithelial cells. Surprisingly, H1N1 and H3N2 viruses recently acquired T145 or N135, respectively, which diminished p85ß binding but apparently not the overall fitness in the human population. Evolutionary analyses revealed covariation of the NS1-p85ß binding phenotype in humans with functional changes at multiple residues in other viral proteins, suggesting an unexplored compensatory or synergistic interplay between phenotypes in vivo Overall, our data provide a resource to understand the consequences of the NS1-p85ß binding spectrum of different influenza viruses and highlight the dynamic evolution of this property in viruses circulating in humans.IMPORTANCE In humans, influenza A viruses are responsible for causing seasonal epidemics and occasional pandemics. These viruses also circulate and evolve in other animal species, creating a reservoir from which novel viruses with distinct properties can emerge. The viral nonstructural protein NS1 is an important host range determinant and virulence factor that exhibits strain-specific interactions with several host factors, although few have been characterized extensively. In the study described here, we comprehensively surveyed the impact of natural and unnatural NS1 variations on the binding of NS1 to host p85ß, a subunit of phosphoinositide 3-kinase that regulates intracellular metabolism and contributes to virus replication and virulence. We define the p85ß-binding site on NS1 and provide a predictive resource to assess this ability of NS1 in viruses from different hosts. Strikingly, we uncover a spectrum of p85ß binding by different NS1 proteins and reveal that viruses evolving in humans have undergone dynamic changes in this NS1 function over the last century.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Evolução Molecular , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/sangue , Influenza Humana/virologia , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Proteínas não Estruturais Virais/genética , Virulência , Replicação Viral
20.
Cell Rep ; 13(7): 1467-1480, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26549460

RESUMO

Dynamic nuclear SUMO modifications play essential roles in orchestrating cellular responses to proteotoxic stress, DNA damage, and DNA virus infection. Here, we describe a non-canonical host SUMOylation response to the nuclear-replicating RNA pathogen, influenza virus, and identify viral RNA polymerase activity as a major contributor to SUMO proteome remodeling. Using quantitative proteomics to compare stress-induced SUMOylation responses, we reveal that influenza virus infection triggers unique re-targeting of SUMO to 63 host proteins involved in transcription, mRNA processing, RNA quality control, and DNA damage repair. This is paralleled by widespread host deSUMOylation. Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes. Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression. Our global characterization of influenza virus-triggered SUMO redistribution provides a proteomic resource to understand host nuclear SUMOylation responses to infection.


Assuntos
Vírus da Influenza A/fisiologia , Sumoilação , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Cães , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Células Madin Darby de Rim Canino , Transporte Proteico , Proteoma/metabolismo , RNA Viral/fisiologia , Ativação Transcricional , Proteínas Supressoras de Tumor/metabolismo , Células Vero
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