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1.
iScience ; : 102908, 2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34337354

RESUMO

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a significant threat to global health. While respiratory aerosols or droplets are considered as the main route of human-to-human transmission, secretions expelled by infected individuals can also contaminate surfaces and objects, potentially creating the risk of fomite-based transmission. Consequently, frequently touched objects such as paper currency and coins have been suspected as potential transmission vehicle. To assess the risk of SARS-CoV-2 transmission by banknotes and coins, we examined the stability of SARS-CoV-2 and bovine coronavirus (BCoV), as surrogate with lower biosafety restrictions, on these different means of payment and developed a touch transfer method to examine transfer efficiency from contaminated surfaces to fingertips. Although we observed prolonged virus stability, our results indicate that transmission of SARS-CoV-2 via contaminated coins and banknotes is unlikely and requires high viral loads and a timely order of specific events.

2.
Nat Commun ; 12(1): 4869, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381043

RESUMO

In COVID-19, immune responses are key in determining disease severity. However, cellular mechanisms at the onset of inflammatory lung injury in SARS-CoV-2 infection, particularly involving endothelial cells, remain ill-defined. Using Syrian hamsters as a model for moderate COVID-19, we conduct a detailed longitudinal analysis of systemic and pulmonary cellular responses, and corroborate it with datasets from COVID-19 patients. Monocyte-derived macrophages in lungs exert the earliest and strongest transcriptional response to infection, including induction of pro-inflammatory genes, while epithelial cells show weak alterations. Without evidence for productive infection, endothelial cells react, depending on cell subtypes, by strong and early expression of anti-viral, pro-inflammatory, and T cell recruiting genes. Recruitment of cytotoxic T cells as well as emergence of IgM antibodies precede viral clearance at day 5 post infection. Investigating SARS-CoV-2 infected Syrian hamsters thus identifies cell type-specific effector functions, providing detailed insights into pathomechanisms of COVID-19 and informing therapeutic strategies.


Assuntos
COVID-19/imunologia , Modelos Animais de Doenças , Células Epiteliais Alveolares/imunologia , Animais , Cricetinae , Citocinas/genética , Citocinas/imunologia , Células Endoteliais/imunologia , Humanos , Imunoglobulina M/imunologia , Inflamação , Pulmão/imunologia , Macrófagos/imunologia , Mesocricetus , Monócitos/imunologia , SARS-CoV-2/imunologia , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia , Receptores Toll-Like/imunologia
3.
Nat Commun ; 12(1): 4584, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321474

RESUMO

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , Antígenos de Diferenciação/genética , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/farmacologia , Antígenos de Diferenciação/metabolismo , Sítios de Ligação , COVID-19/virologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Interferon beta/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos
4.
J Clin Invest ; 131(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34061776

RESUMO

Autoantibodies against IFN-α and IFN-ω (type I IFNs) were recently reported as causative for severe COVID-19 in the general population. Autoantibodies against IFN-α and IFN-ω are present in almost all patients with autoimmune polyendocrine syndrome type 1 (APS-1) caused by biallelic deleterious or heterozygous dominant mutations in AIRE. We therefore hypothesized that autoantibodies against type I IFNs also predispose patients with APS-1 to severe COVID-19. We prospectively studied 6 patients with APS-1 between April 1, 2020 and April 1, 2021. Biobanked pre-COVID-19 sera of APS-1 subjects were tested for neutralizing autoantibodies against IFN-α and IFN-ω. The ability of the patients' sera to block recombinant human IFN-α and IFN-ω was assessed by assays quantifying phosphorylation of signal transducer and activator of transcription 1 (STAT1) as well as infection-based IFN-neutralization assays. We describe 4 patients with APS-1 and preexisting high titers of neutralizing autoantibodies against IFN-α and IFN-ω who contracted SARS-CoV-2, yet developed only mild symptoms of COVID-19. None of the patients developed dyspnea, oxygen requirement, or high temperature. All infected patients with APS-1 were females and younger than 26 years of age. Clinical penetrance of neutralizing autoantibodies against type I IFNs for severe COVID-19 is not complete.


Assuntos
Autoanticorpos/imunologia , COVID-19/complicações , COVID-19/imunologia , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/imunologia , Poliendocrinopatias Autoimunes/complicações , Poliendocrinopatias Autoimunes/imunologia , SARS-CoV-2 , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Autoanticorpos/sangue , Feminino , Humanos , Técnicas In Vitro , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/imunologia , Masculino , Poliendocrinopatias Autoimunes/genética , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença , Fatores de Transcrição/genética , Replicação Viral/imunologia , Adulto Jovem
5.
Life Sci Alliance ; 4(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34078739

RESUMO

Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3 Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.

6.
AIDS ; 35(13): 2105-2117, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34155151

RESUMO

OBJECTIVE: The cervical mucus plugs are enriched with proteins of known immunological functions. We aimed to characterize the anti-HIV-1 activity of the cervical mucus plugs against a panel of different HIV-1 strains in the contexts of cell-free and cell-associated virus. DESIGN: A cohort of consenting HIV-1-negative and HIV-1-positive pregnant women in labour was recruited from Mthatha General Hospital in the Eastern Cape province of South Africa, from whom the cervical mucus plugs were collected in 6 M guanidinium chloride with protease inhibitors and transported to our laboratories at -80 °C. METHODS: Samples were centrifuged to remove insoluble material and dialysed before freeze--drying and subjecting them to the cell viability assays. The antiviral activities of the samples were studied using luminometric reporter assays and flow cytometry. Time-of-addition and BlaM-Vpr virus-cell fusion assays were used to pin-point the antiviral mechanisms of the cervical mucus plugs, before proteomic profiling using liquid chromatography-tandem mass spectrometry. RESULTS: The proteinaceous fraction of the cervical mucus plugs exhibited anti-HIV-1 activity with inter-individual variations and some degree of specificity among different HIV-1 strains. Cell-associated HIV-1 was less susceptible to inhibition by the potent samples whenever compared with the cell-free HIV-1. The samples with high antiviral potency exhibited a distinct proteomic profile when compared with the less potent samples. CONCLUSION: The crude cervical mucus plugs exhibit anti-HIV-1 activity, which is defined by a specific proteomic profile.


Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Muco do Colo Uterino , Feminino , Humanos , Gravidez , Proteômica
7.
Nat Biotechnol ; 39(6): 705-716, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33361824

RESUMO

In coronavirus disease 2019 (COVID-19), hypertension and cardiovascular diseases are major risk factors for critical disease progression. However, the underlying causes and the effects of the main anti-hypertensive therapies-angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)-remain unclear. Combining clinical data (n = 144) and single-cell sequencing data of airway samples (n = 48) with in vitro experiments, we observed a distinct inflammatory predisposition of immune cells in patients with hypertension that correlated with critical COVID-19 progression. ACEI treatment was associated with dampened COVID-19-related hyperinflammation and with increased cell intrinsic antiviral responses, whereas ARB treatment related to enhanced epithelial-immune cell interactions. Macrophages and neutrophils of patients with hypertension, in particular under ARB treatment, exhibited higher expression of the pro-inflammatory cytokines CCL3 and CCL4 and the chemokine receptor CCR1. Although the limited size of our cohort does not allow us to establish clinical efficacy, our data suggest that the clinical benefits of ACEI treatment in patients with COVID-19 who have hypertension warrant further investigation.


Assuntos
COVID-19/tratamento farmacológico , Quimiocina CCL3/genética , Quimiocina CCL4/genética , Hipertensão/tratamento farmacológico , Receptores CCR1/genética , Adulto , Antagonistas de Receptores de Angiotensina/administração & dosagem , Antagonistas de Receptores de Angiotensina/efeitos adversos , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/efeitos adversos , COVID-19/complicações , COVID-19/genética , COVID-19/virologia , Progressão da Doença , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertensão/complicações , Hipertensão/genética , Hipertensão/patologia , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/virologia , Masculino , Pessoa de Meia-Idade , RNA-Seq , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/patologia , Sistema Respiratório/virologia , Fatores de Risco , SARS-CoV-2/patogenicidade , Análise de Célula Única
8.
Cell ; 182(6): 1419-1440.e23, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32810438

RESUMO

Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.


Assuntos
Infecções por Coronavirus/imunologia , Células Mieloides/imunologia , Mielopoese , Pneumonia Viral/imunologia , Adulto , Idoso , Antígenos CD11/genética , Antígenos CD11/metabolismo , COVID-19 , Células Cultivadas , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/citologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Proteoma/genética , Proteoma/metabolismo , Proteômica , Análise de Célula Única
9.
Proc Natl Acad Sci U S A ; 117(32): 19475-19486, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709741

RESUMO

The DNA sensor cGAS catalyzes the production of the cyclic dinucleotide cGAMP, resulting in type I interferon responses. We addressed the functionality of cGAS-mediated DNA sensing in human and murine T cells. Activated primary CD4+ T cells expressed cGAS and responded to plasmid DNA by upregulation of ISGs and release of bioactive interferon. In mouse T cells, cGAS KO ablated sensing of plasmid DNA, and TREX1 KO enabled cells to sense short immunostimulatory DNA. Expression of IFIT1 and MX2 was downregulated and upregulated in cGAS KO and TREX1 KO T cell lines, respectively, compared to parental cells. Despite their intact cGAS sensing pathway, human CD4+ T cells failed to mount a reverse transcriptase (RT) inhibitor-sensitive immune response following HIV-1 infection. In contrast, infection of human T cells with HSV-1 that is functionally deficient for the cGAS antagonist pUL41 (HSV-1ΔUL41N) resulted in a cGAS-dependent type I interferon response. In accordance with our results in primary CD4+ T cells, plasmid challenge or HSV-1ΔUL41N inoculation of T cell lines provoked an entirely cGAS-dependent type I interferon response, including IRF3 phosphorylation and expression of ISGs. In contrast, no RT-dependent interferon response was detected following transduction of T cell lines with VSV-G-pseudotyped lentiviral or gammaretroviral particles. Together, T cells are capable to raise a cGAS-dependent cell-intrinsic response to both plasmid DNA challenge or inoculation with HSV-1ΔUL41N. However, HIV-1 infection does not appear to trigger cGAS-mediated sensing of viral DNA in T cells, possibly by revealing viral DNA of insufficient quantity, length, and/or accessibility to cGAS.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , DNA Viral/fisiologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Herpesvirus Humano 1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Camundongos , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Especificidade da Espécie , Replicação Viral
10.
Nat Biotechnol ; 38(8): 970-979, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32591762

RESUMO

To investigate the immune response and mechanisms associated with severe coronavirus disease 2019 (COVID-19), we performed single-cell RNA sequencing on nasopharyngeal and bronchial samples from 19 clinically well-characterized patients with moderate or critical disease and from five healthy controls. We identified airway epithelial cell types and states vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In patients with COVID-19, epithelial cells showed an average three-fold increase in expression of the SARS-CoV-2 entry receptor ACE2, which correlated with interferon signals by immune cells. Compared to moderate cases, critical cases exhibited stronger interactions between epithelial and immune cells, as indicated by ligand-receptor expression profiles, and activated immune cells, including inflammatory macrophages expressing CCL2, CCL3, CCL20, CXCL1, CXCL3, CXCL10, IL8, IL1B and TNF. The transcriptional differences in critical cases compared to moderate cases likely contribute to clinical observations of heightened inflammatory tissue damage, lung injury and respiratory failure. Our data suggest that pharmacologic inhibition of the CCR1 and/or CCR5 pathways might suppress immune hyperactivation in critical COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Pneumonia Viral/patologia , Pneumonia Viral/fisiopatologia , Sistema Respiratório/patologia , Análise de Célula Única , Transcriptoma , Adulto , Idoso , Enzima de Conversão de Angiotensina 2 , Líquido da Lavagem Broncoalveolar/virologia , COVID-19 , Comunicação Celular , Diferenciação Celular , Infecções por Coronavirus/virologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Humanos , Sistema Imunitário/patologia , Inflamação/imunologia , Inflamação/patologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Peptidil Dipeptidase A/genética , Pneumonia Viral/virologia , Sistema Respiratório/imunologia , Sistema Respiratório/virologia , Índice de Gravidade de Doença
11.
Immunity ; 52(5): 719-721, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433941

RESUMO

Cytosolic DNA detection via the DNA sensor cGAS initiates a major cell-intrinsic response to infection and malignancies. In this issue of Immunity, Zhou et al. (2020) report that the catalytic product of cGAS, cGAMP, can alert bystander cells over large distances through its cell-to-cell transmission via volume-regulated anion channels.


Assuntos
Antivirais , Interferons , Ânions , Imunidade Inata , Nucleotídeos Cíclicos , Nucleotidiltransferases
12.
J Virol ; 94(2)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666384

RESUMO

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t 1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.


Assuntos
Bacteriocinas/farmacologia , Microdomínios da Membrana/metabolismo , Viroses/metabolismo , Vírus/metabolismo , Aedes , Animais , Linhagem Celular , Microdomínios da Membrana/virologia , Fosfatidiletanolaminas/metabolismo , Viroses/tratamento farmacológico
13.
J Virol ; 93(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31597782

RESUMO

When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface.IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Genótipo , Células HEK293 , Infecções por HIV/virologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Interferon-alfa , Proteínas de Membrana/genética , Pirazóis/farmacologia , Linfócitos T/virologia , Vírion/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo
14.
Mol Cell Proteomics ; 18(12): 2401-2417, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570497

RESUMO

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus Uukuniemi/fisiologia , Animais , Antivirais/farmacologia , Infecções por Bunyaviridae/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Espectrometria de Massas , Proteômica , Piridinas/farmacologia , Quinolinas/farmacologia , Interferência de RNA , Vírus de RNA/fisiologia , Vírus Uukuniemi/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Replicação Viral
15.
PLoS Pathog ; 14(8): e1007269, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30125328

RESUMO

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.


Assuntos
Linfócitos T CD4-Positivos/virologia , Produtos do Gene nef/fisiologia , HIV-1/fisiologia , Tecido Linfoide/virologia , Proteínas de Membrana/metabolismo , Replicação Viral/genética , Animais , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Colobus/virologia , Células HEK293 , Humanos , Células Jurkat , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Vírus da Imunodeficiência Símia/genética
16.
J Infect Dis ; 218(9): 1507-1510, 2018 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-29917109

RESUMO

Despite increasing clinical relevance of Chikungunya virus (CHIKV) infection, caused by a rapidly emerging pathogen, recommended guidelines for its inactivation do not exist. In this study, we investigated the susceptibility of CHIKV to inactivation by heat and commercially available hand, surface, and World Health Organization-recommended disinfectants to define CHIKV prevention protocols for healthcare systems.


Assuntos
Febre de Chikungunya/prevenção & controle , Vírus Chikungunya/efeitos dos fármacos , Desinfetantes/farmacologia , Linhagem Celular , Células HEK293 , Temperatura Alta , Humanos , Organização Mundial da Saúde
17.
J Virol ; 92(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29743357

RESUMO

Cellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent human immunodeficiency virus type 1 (HIV-1) virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce the particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered the fact that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient, for 90K-mediated antiviral activity. Rather, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single "humanizing" amino acid exchange in the BTB (broad-complex, tramtrack, and bric-à-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue the antiviral activity of a shortened version of rhesus macaque 90K, but not that of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domains of 90K from rhesus macaques and humans point toward a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein.IMPORTANCE The cellular 90K protein has been shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear smaller amounts of the HIV-1 Env glycoprotein, which is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but, surprisingly, not from rhesus macaques, inhibit HIV-1. The introduction of a single amino acid exchange into a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.


Assuntos
Antígenos de Neoplasias/farmacologia , Antivirais/farmacologia , Biomarcadores Tumorais/farmacologia , Proteínas de Transporte/farmacologia , Glicoproteínas/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/química , Biomarcadores Tumorais/química , Proteínas de Transporte/química , Produtos do Gene env/metabolismo , Glicoproteínas/química , Infecções por HIV/virologia , Humanos , Macaca mulatta , Conformação Proteica , Homologia de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Especificidade da Espécie , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
18.
Retrovirology ; 14(1): 48, 2017 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-29047401

RESUMO

The authors wish to apologize for an error within the scale bar of the microarray heatmap in Additional File 5 of the supplementary information. Two values were incorrectly displayed on the scale bar (11 instead of 10 and 13 instead of 12).

20.
Retrovirology ; 14(1): 34, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28569216

RESUMO

BACKGROUND: Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. RESULTS: In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. CONCLUSIONS: We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.


Assuntos
Vetores Genéticos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/virologia , Lentivirus/genética , Animais , Proteínas do Capsídeo/genética , Proteínas de Transporte/genética , Linhagem Celular , Ciclofilina A/metabolismo , Ciclosporina/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Lentivirus/fisiologia , Leupeptinas/farmacologia , Camundongos , Transcrição Reversa/efeitos dos fármacos , Transdução Genética , Integração Viral/efeitos dos fármacos , Internalização do Vírus
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