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1.
EBioMedicine ; 70: 103495, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34304047

RESUMO

BACKGROUND: Children are underrepresented in the COVID-19 pandemic and often experience milder disease than adolescents and adults. Reduced severity is possibly due to recent and more frequent seasonal human coronaviruses (HCoV) infections. We assessed the seroprevalence of SARS-CoV-2 and seasonal HCoV specific antibodies in a large cohort in north-eastern France. METHODS: In this cross-sectional seroprevalence study, serum samples were collected from children and adults requiring hospital admission for non-COVID-19 between February and August 2020. Antibody responses to SARS-CoV-2 and seasonal HCoV (229E, HKU1, NL63, OC43) were assessed using a bead-based multiplex assay, Luciferase-Linked ImmunoSorbent Assay, and a pseudotype neutralisation assay. FINDINGS: In 2,408 individuals, seroprevalence of SARS-CoV-2-specific antibodies was 7-8% with three different immunoassays. Antibody levels to seasonal HCoV increased substantially up to the age of 10. Antibody responses in SARS-CoV-2 seropositive individuals were lowest in adults 18-30 years. In SARS-CoV-2 seronegative individuals, we observed cross-reactivity between antibodies to the four HCoV and SARS-CoV-2 Spike. In contrast to other antibodies to SARS-CoV-2, specific antibodies to sub-unit 2 of Spike (S2) in seronegative samples were highest in children. Upon infection with SARS-CoV-2, antibody levels to Spike of betacoronavirus OC43 increased across the whole age spectrum. No SARS-CoV-2 seropositive individuals with low levels of antibodies to seasonal HCoV were observed. INTERPRETATION: Our findings underline significant cross-reactivity between antibodies to SARS-CoV-2 and seasonal HCoV, but provide no significant evidence for cross-protective immunity to SARS-CoV-2 infection due to a recent seasonal HCoV infection. In particular, across all age groups we did not observe SARS-CoV-2 infected individuals with low levels of antibodies to seasonal HCoV. FUNDING: This work was supported by the « URGENCE COVID-19 ¼ fundraising campaign of Institut Pasteur, by the French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (Grant No. ANR-10-LABX-62-IBEID), and by the REACTing (Research & Action Emerging Infectious Diseases), and by the RECOVER project funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No. 101003589, and by a grant from LabEx IBEID (ANR-10-LABX-62-IBEID).


Assuntos
COVID-19/imunologia , Imunidade Humoral/imunologia , SARS-CoV-2/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/imunologia , Criança , Pré-Escolar , Ensaios Clínicos como Assunto , Reações Cruzadas/imunologia , Estudos Transversais , Feminino , França , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Pandemias/prevenção & controle , Estações do Ano , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
2.
Nat Commun ; 12(1): 3025, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021152

RESUMO

Assessment of the cumulative incidence of SARS-CoV-2 infections is critical for monitoring the course and extent of the COVID-19 epidemic. Here, we report estimated seroprevalence in the French population and the proportion of infected individuals who developed neutralising antibodies at three points throughout the first epidemic wave. Testing 11,000 residual specimens for anti-SARS-CoV-2 IgG and neutralising antibodies, we find nationwide seroprevalence of 0.41% (95% CI: 0.05-0.88) mid-March, 4.14% (95% CI: 3.31-4.99) mid-April and 4.93% (95% CI: 4.02-5.89) mid-May 2020. Approximately 70% of seropositive individuals have detectable neutralising antibodies. Infection fatality rate is 0.84% (95% CI: 0.70-1.03) and increases exponentially with age. These results confirm that the nationwide lockdown substantially curbed transmission and that the vast majority of the French population remained susceptible to SARS-CoV-2 in May 2020. Our study shows the progression of the first epidemic wave and provides a framework to inform the ongoing public health response as viral transmission continues globally.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , COVID-19/epidemiologia , COVID-19/virologia , Criança , Pré-Escolar , Epidemias , Feminino , França/epidemiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Prevalência , SARS-CoV-2/fisiologia , Estudos Soroepidemiológicos , Adulto Jovem
3.
Euro Surveill ; 26(15)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33860747

RESUMO

BackgroundChildren's role in SARS-CoV-2 epidemiology remains unclear. We investigated an initially unnoticed SARS-CoV-2 outbreak linked to schools in northern France, beginning as early as mid-January 2020.AimsThis retrospective observational study documents the extent of SARS-CoV-2 transmission, linked to an affected high school (n = 664 participants) and primary schools (n = 1,340 study participants), in the context of unsuspected SARS-CoV-2 circulation and limited control measures.MethodsBetween 30 March and 30 April 2020, all school staff, as well as pupils and their parents and relatives were invited for SARS-CoV-2 antibody testing and to complete a questionnaire covering symptom history since 13 January 2020.ResultsIn the high school, infection attack rates were 38.1% (91/239), 43.4% (23/53), and 59.3% (16/27), in pupils, teachers, and non-teaching staff respectively vs 10.1% (23/228) and 12.0% (14/117) in the pupils' parents and relatives (p < 0.001). Among the six primary schools, three children attending separate schools at the outbreak start, while symptomatic, might have introduced SARS-CoV-2 there, but symptomatic secondary cases related to them could not be definitely identified. In the primary schools overall, antibody prevalence in pupils sharing classes with symptomatic cases was higher than in pupils from other classes: 15/65 (23.1%) vs 30/445 (6.7%) (p < 0.001). Among 46 SARS-CoV-2 seropositive pupils < 12 years old, 20 were asymptomatic. Whether past HKU1 and OC43 seasonal coronavirus infection protected against SARS-CoV-2 infection in 6-11 year olds could not be inferred.ConclusionsViral circulation can occur in high and primary schools so keeping them open requires consideration of appropriate control measures and enhanced surveillance.


Assuntos
COVID-19 , Criança , Estudos de Coortes , França/epidemiologia , Humanos , Estudos Retrospectivos , SARS-CoV-2 , Instituições Acadêmicas
5.
Lancet Microbe ; 2(2): e60-e69, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33521709

RESUMO

Background: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces an antibody response targeting multiple antigens that changes over time. This study aims to take advantage of this complexity to develop more accurate serological diagnostics. Methods: A multiplex serological assay was developed to measure IgG and IgM antibody responses to seven SARS-CoV-2 spike or nucleoprotein antigens, two antigens for the nucleoproteins of the 229E and NL63 seasonal coronaviruses, and three non-coronavirus antigens. Antibodies were measured in serum samples collected up to 39 days after symptom onset from 215 adults in four French hospitals (53 patients and 162 health-care workers) with quantitative RT-PCR-confirmed SARS-CoV-2 infection, and negative control serum samples collected from healthy adult blood donors before the start of the SARS-CoV-2 epidemic (335 samples from France, Thailand, and Peru). Machine learning classifiers were trained with the multiplex data to classify individuals with previous SARS-CoV-2 infection, with the best classification performance displayed by a random forests algorithm. A Bayesian mathematical model of antibody kinetics informed by prior information from other coronaviruses was used to estimate time-varying antibody responses and assess the sensitivity and classification performance of serological diagnostics during the first year following symptom onset. A statistical estimator is presented that can provide estimates of seroprevalence in very low-transmission settings. Findings: IgG antibody responses to trimeric spike protein (Stri) identified individuals with previous SARS-CoV-2 infection with 91·6% (95% CI 87·5-94·5) sensitivity and 99·1% (97·4-99·7) specificity. Using a serological signature of IgG and IgM to multiple antigens, it was possible to identify infected individuals with 98·8% (96·5-99·6) sensitivity and 99·3% (97·6-99·8) specificity. Informed by existing data from other coronaviruses, we estimate that 1 year after infection, a monoplex assay with optimal anti-Stri IgG cutoff has 88·7% (95% credible interval 63·4-97·4) sensitivity and that a four-antigen multiplex assay can increase sensitivity to 96·4% (80·9-100·0). When applied to population-level serological surveys, statistical analysis of multiplex data allows estimation of seroprevalence levels less than 2%, below the false-positivity rate of many other assays. Interpretation: Serological signatures based on antibody responses to multiple antigens can provide accurate and robust serological classification of individuals with previous SARS-CoV-2 infection. This provides potential solutions to two pressing challenges for SARS-CoV-2 serological surveillance: classifying individuals who were infected more than 6 months ago and measuring seroprevalence in serological surveys in very low-transmission settings. Funding: European Research Council. Fondation pour la Recherche Médicale. Institut Pasteur Task Force COVID-19.

6.
Nucleic Acids Res ; 48(18): 10428-10440, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960265

RESUMO

Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus.


Assuntos
Exorribonucleases/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Proteínas Nucleares/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Linhagem Celular , Histonas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno , RNA Viral/genética , Ribonucleoproteínas/genética , Transcrição Genética/genética , Proteínas Virais/genética , Replicação Viral/genética
7.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética , SARS-CoV-2
8.
Sci Transl Med ; 12(559)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32817357

RESUMO

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19 , Teste para COVID-19 , Estudos de Coortes , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , França/epidemiologia , Voluntários Saudáveis , Humanos , Imunoprecipitação/métodos , Luciferases , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , SARS-CoV-2 , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Pesquisa Médica Translacional , Adulto Jovem
9.
mBio ; 11(2)2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265326

RESUMO

The multifunctional nature of viral proteins is essentially driven by posttranslational modifications (PTMs) and is key for the successful outcome of infection. For influenza A viruses (IAVs), a composite pattern of PTMs regulates the activity of viral proteins. However, almost none are known that target the PB2 replication protein, except for inducing its degradation. We show here that PB2 undergoes a nonproteolytic ubiquitination during infection. We identified E3 ubiquitin ligases catalyzing this ubiquitination as two multicomponent RING-E3 ligases based on cullin 4 (CRL4s), which are both contributing to the levels of ubiquitinated forms of PB2 in infected cells. The CRL4 E3 ligase activity is required for the normal progression of the viral cycle and for maximal virion production, indicating that the CRL4s mediate a ubiquitin signaling that promotes infection. The CRL4s are recruiting PB2 through an unconventional bimodal interaction with both the DDB1 adaptor and DCAF substrate receptors. While able to bind to PB2 when engaged in the viral polymerase complex, the CRL4 factors do not alter transcription and replication of the viral segments during infection. CRL4 ligases catalyze different patterns of lysine ubiquitination on PB2. Recombinant viruses mutated in the targeted lysines showed attenuated viral production, suggesting that CRL4-mediated ubiquitination of PB2 contributes to IAV infection. We identified K29-linked ubiquitin chains as main components of the nonproteolytic PB2 ubiquitination mediated by the CRL4s, providing the first example of the role of this atypical ubiquitin linkage in the regulation of a viral infection.IMPORTANCE Successful infection by influenza A virus, a pathogen of major public health importance, involves fine regulation of the multiple functions of the viral proteins, which often relies on post-translational modifications (PTMs). The PB2 protein of influenza A viruses is essential for viral replication and a key determinant of host range. While PTMs of PB2 inducing its degradation have been identified, here we show that PB2 undergoes a regulating PTM signaling detected during infection, based on an atypical K29-linked ubiquitination and mediated by two multicomponent E3 ubiquitin ligases. Recombinant viruses impaired for CRL4-mediated ubiquitination are attenuated, indicating that ubiquitination of PB2 is necessary for an optimal influenza A virus infection. The CRL4 E3 ligases are required for normal viral cycle progression and for maximal virion production. Consequently, they represent potential candidate host factors for antiviral targets.


Assuntos
Proteínas Culina/metabolismo , Vírus da Influenza A/química , Provírus/enzimologia , RNA Polimerase Dependente de RNA/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Virais/química , Replicação Viral , Células A549 , Proteínas Culina/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/fisiologia , Processamento de Proteína Pós-Traducional
10.
Cell Rep ; 30(5): 1570-1584.e6, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32023470

RESUMO

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses.


Assuntos
Cisteína Endopeptidases/metabolismo , Proteína DEAD-box 58/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/metabolismo , Células A549 , Animais , Citosol/metabolismo , Enzimas Desubiquitinantes/metabolismo , Cães , Deleção de Genes , Células HEK293 , Humanos , Influenza Humana , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Células Madin Darby de Rim Canino , Masculino , Membranas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica
11.
Nat Commun ; 10(1): 3907, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467278

RESUMO

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.


Assuntos
Bioensaio/métodos , Mapeamento de Interação de Proteínas/métodos , Proteoma/análise , Bases de Dados de Proteínas , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Mapas de Interação de Proteínas , Proteínas/metabolismo , Proteômica/métodos , Técnicas do Sistema de Duplo-Híbrido
12.
Cell Rep ; 26(7): 1800-1814.e5, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759391

RESUMO

The mechanisms that regulate envelopment of HCV and other viruses that bud intracellularly and/or lack late-domain motifs are largely unknown. We reported that K63 polyubiquitination of the HCV nonstructural (NS) 2 protein mediates HRS (ESCRT-0 component) binding and envelopment. Nevertheless, the ubiquitin signaling that governs NS2 ubiquitination remained unknown. Here, we map the NS2 interactome with the ubiquitin proteasome system (UPS) via mammalian cell-based screens. NS2 interacts with E3 ligases, deubiquitinases, and ligase regulators, some of which are candidate proviral or antiviral factors. MARCH8, a RING-finger E3 ligase, catalyzes K63-linked NS2 polyubiquitination in vitro and in HCV-infected cells. MARCH8 is required for infection with HCV, dengue, and Zika viruses and specifically mediates HCV envelopment. Our data reveal regulation of HCV envelopment via ubiquitin signaling and both a viral protein substrate and a ubiquitin K63-linkage of the understudied MARCH8, with potential implications for cell biology, virology, and host-targeted antiviral design.


Assuntos
Hepacivirus/metabolismo , Hepatite C/virologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Células HEK293 , Hepacivirus/patogenicidade , Hepatite C/genética , Hepatite C/metabolismo , Humanos , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
14.
Oncotarget ; 9(17): 13102-13115, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29568343

RESUMO

The SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) is a broadly expressed adaptor associated with the control of actin-polymerization, cell migration, and oncogenesis. After activation of different receptors at the cell surface, this dimeric protein serves as a platform for assembling other adaptors such as FYB and some SRC family kinase members, although these mechanisms are still poorly understood. The goal of this study is to map the SKAP2 interactome and characterize which domains or binding motifs are involved in these interactions. This is a prerequisite to finely analyze how these pathways are integrated in the cell machinery and to study their role in cancer and other human diseases when this network of interactions is perturbed. In this work, the domain and the binding motif of fourteen proteins interacting with SKAP2 were precisely defined and a new interactor, FAM102A was discovered. Herein, a fine-tuning between the binding of SRC kinases and their activation was identified. This last process, which depends on SKAP2 dimerization, indirectly affects the binding of FYB protein. Analysis of conformational changes associated with activation/inhibition of SRC family members, presently limited to their effect on kinase activity, is extended to their interactive network, which paves the way for therapeutic development.

15.
mSphere ; 2(6)2017.
Artigo em Inglês | MEDLINE | ID: mdl-29202037

RESUMO

The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions (PPIs) on the basis of comparative interactomics of multiple strains is an effective strategy to highlight correlations between host proteome hijacking and biological or pathogenic traits. Here, we developed an interactomic pipeline to deliver high-confidence comparative maps of PPIs between a given pathogen and the human ubiquitin proteasome system (UPS). This subarray of the human proteome represents a range of essential cellular functions and promiscuous targets for many viruses. The screening pipeline was applied to the influenza A virus (IAV) PB2 polymerase proteins of five strains representing different levels of virulence in humans. An extensive PB2-UPS interplay has been detected that recapitulates the evolution of IAVs in humans. Functional validation with several IAV strains, including the seasonal H1N1pdm09 and H3N2 viruses, confirmed the biological relevance of most identified UPS factors and revealed strain-independent and strain-specific effects of UPS factor invalidation on IAV infection. This strategy is applicable to proteins from any other virus or pathogen, providing a valuable resource with which to explore the UPS-pathogen interplay and its relationship with pathogenicity. IMPORTANCE Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus's adaptation to humans. This suggests that UPS hijacking underlies the efficient infection of humans and can be used as an indicator for evaluation of the potential of avian IAVs to infect humans. Several UPS factors were found to be necessary for infection with circulating IAV strains, pointing to potential targets for therapeutic approaches.

16.
FEBS J ; 284(19): 3171-3201, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28786561

RESUMO

Protein ubiquitination and its reverse reaction, deubiquitination, regulate protein stability, protein binding activity, and their subcellular localization. These reactions are catalyzed by the enzymes E1, E2, and E3 ubiquitin (Ub) ligases and deubiquitinases (DUBs). The Ub-proteasome system (UPS) is targeted by viruses for the sake of their replication and to escape host immune response. To identify novel partners of human papillomavirus 16 (HPV16) E6 and E7 proteins, we assembled and screened a library of 590 cDNAs related to the UPS by using the Gaussia princeps luciferase protein complementation assay. HPV16 E6 was found to bind to the homology to E6AP C terminus-type Ub ligase (E6AP), three really interesting new gene (RING)-type Ub ligases (MGRN1, LNX3, LNX4), and the DUB Ub-specific protease 15 (USP15). Except for E6AP, the binding of UPS factors did not require the LxxLL-binding pocket of HPV16 E6. LNX3 bound preferentially to all high-risk mucosal HPV E6 tested, whereas LNX4 bound specifically to HPV16 E6. HPV16 E7 was found to bind to several broad-complex tramtrack and bric-a-brac domain-containing proteins (such as TNFAIP1/KCTD13) that are potential substrate adaptors of Cullin 3-RING Ub ligases, to RING-type Ub ligases implicated in innate immunity (RNF135, TRIM32, TRAF2, TRAF5), to the substrate adaptor DCAF15 of Cullin 4-RING Ub ligase and to some DUBs (USP29, USP33). The binding to UPS factors did not require the LxCxE motif but rather the C-terminal region of HPV16 E7 protein. The identified UPS factors interacted with most of E7 proteins across different HPV types. This study establishes a strategy for the rapid identification of interactions between host or pathogen proteins and the human ubiquitination system.


Assuntos
Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/genética , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina/genética , Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Biologia Computacional , Regulação da Expressão Gênica , Genes Reporter , Papillomavirus Humano 16/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Anotação de Sequência Molecular , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Replicação Viral
17.
Oncotarget ; 8(28): 46163-46176, 2017 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-28521287

RESUMO

High-risk human papillomaviruses are the etiological agents of cervical cancer and HPV16 is the most oncogenic genotype. Immortalization and transformation of infected cells requires the overexpression of the two viral oncoproteins E6 and E7 following HPV DNA integration into the host cell genome. Integration often leads to the loss of the E2 open reading frame and the corresponding protein can no longer act as a transcriptional repressor on p97 promoter. Recently, it has been proposed that long control region methylation also contributes to the regulation of E6/E7 expression.To determine which epigenetic mechanism is involved in HPV16 early gene regulation, 5-aza-2'-deoxycytidine was used to demethylate Ca Ski and SiHa cell DNA. Decreased expression of E6 mRNA and protein levels was observed in both cell lines in an E2-independent manner. E6 repression was accompanied by neither a modification of the main cellular transcription factor expression involved in long control region regulation, nor by a modification of the E6 mRNA splicing pattern. In contrast, a pronounced upregulation of miR-375, known to destabilize HPV16 early viral mRNA, was observed. Finally, the use of miR-375 inhibitor definitively proved the involvement of miR-375 in E6 repression. These results highlight that cellular DNA methylation modulates HPV16 early gene expression and support a role for epigenetic events in high-risk HPV associated-carcinogenesis.


Assuntos
Azacitidina/análogos & derivados , Papillomavirus Humano 16/genética , MicroRNAs/genética , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/tratamento farmacológico , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , Metilação de DNA , Decitabina , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/genética , Proteínas Repressoras/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero/genética
18.
Virologie (Montrouge) ; 20(6): 302-320, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32187967

RESUMO

Influenza viruses are segmented negative-sense RNA viruses whose RNA dependant RNA polymerase (RdRp) multiple activities multiple activities are central for the viral life cycle. The RdRp is composed of three subunits, PB1, PB2 and PA. It binds to the extremities of each vRNA segments encapsidated with multiple copies of the Nucleoprotein (NP), altogether constituting the viral ribonucleoprotein (vRNP). The RdRp performs both vRNA transcription and replication in the context of vRNP in the nuclei of infected cells. The temporal regulation of RdRp-associated activities is essential for the successful completion of the virus life cycle, but its understanding has been limited by the lack of structural information about the polymerase complex. The atomic-resolution of polymerase complexes from influenza virus type A, type B and type C came out in the past two years.We compile here the data provided by the near-concomitant resolution of several influenza polymerase crystal structures. We will highlight how structural information can contribute to our understanding of the interactions between the RdRp and viral or host factors.

19.
Virologie (Montrouge) ; 20(6): 32-48, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32187969

RESUMO

Influenza viruses are segmented negative-sense RNA viruses whose RNA dependant RNA polymerase (RdRp) multiple activities are central for the viral life cycle. The RdRp is composed of three subunits, PB1, PB2 and PA. It binds to the extremities of each vRNA segments encapsidated with multiple copies of the Nucleoprotein (NP), altogether constituting the viral ribonucleoproteins (vRNPs). The RdRp performs both vRNA transcription and replication in the context of vRNP in the nuclei of infected cells. The temporal regulation of RdRp-associated activities is essential for the successful completion of the virus life cycle, but its understanding has been limited by the lack of structural information about the polymerase complex. The atomic-resolution of polymerase complexes from influenza virus type A, type B and type C came out in the past two years. We compile here the data provided by the near-concomitant resolution of several influenza polymerase crystal structures. We will highlight how structural information can contribute to our understanding of the interactions between the RdRp and viral or host factors.

20.
PLoS Pathog ; 10(6): e1004164, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24945353

RESUMO

Influenza A viruses are major pathogens in humans and in animals, whose genome consists of eight single-stranded RNA segments of negative polarity. Viral mRNAs are synthesized by the viral RNA-dependent RNA polymerase in the nucleus of infected cells, in close association with the cellular transcriptional machinery. Two proteins essential for viral multiplication, the exportin NS2/NEP and the ion channel protein M2, are produced by splicing of the NS1 and M1 mRNAs, respectively. Here we identify two human spliceosomal factors, RED and SMU1, that control the expression of NS2/NEP and are required for efficient viral multiplication. We provide several lines of evidence that in infected cells, the hetero-trimeric viral polymerase recruits a complex formed by RED and SMU1 through interaction with its PB2 and PB1 subunits. We demonstrate that the splicing of the NS1 viral mRNA is specifically affected in cells depleted of RED or SMU1, leading to a decreased production of the spliced mRNA species NS2, and to a reduced NS2/NS1 protein ratio. In agreement with the exportin function of NS2, these defects impair the transport of newly synthesized viral ribonucleoproteins from the nucleus to the cytoplasm, and strongly reduce the production of infectious influenza virions. Overall, our results unravel a new mechanism of viral subversion of the cellular splicing machinery, by establishing that the human splicing factors RED and SMU1 act jointly as key regulators of influenza virus gene expression. In addition, our data point to a central role of the viral RNA polymerase in coupling transcription and alternative splicing of the viral mRNAs.


Assuntos
Processamento Alternativo , Proteínas Cromossômicas não Histona/metabolismo , Citocinas/metabolismo , Vírus da Influenza A/fisiologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Citocinas/antagonistas & inibidores , Citocinas/química , Citocinas/genética , Inativação Gênica , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/enzimologia , Carioferinas/genética , Carioferinas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Spliceossomos/enzimologia , Spliceossomos/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Replicação Viral
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