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1.
Elife ; 92020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32149608

RESUMO

Proliferating cells must coordinate central metabolism with the cell cycle. How central energy metabolism regulates bacterial cell cycle functions is not well understood. Our forward genetic selection unearthed the Krebs cycle enzyme citrate synthase (CitA) as a checkpoint regulator controlling the G1→S transition in the polarized alpha-proteobacterium Caulobacter crescentus, a model for cell cycle regulation and asymmetric cell division. We find that loss of CitA promotes the accumulation of active CtrA, an essential cell cycle transcriptional regulator that maintains cells in G1-phase, provided that the (p)ppGpp alarmone is present. The enzymatic activity of CitA is dispensable for CtrA control, and functional citrate synthase paralogs cannot replace CitA in promoting S-phase entry. Our evidence suggests that CitA was appropriated specifically to function as a moonlighting enzyme to link central energy metabolism with S-phase entry. Control of the G1-phase by a central metabolic enzyme may be a common mechanism of cellular regulation.


Assuntos
Caulobacter crescentus/fisiologia , Pontos de Checagem do Ciclo Celular , Citrato (si)-Sintase/metabolismo , Fase G1 , Fase S , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/enzimologia , Caulobacter crescentus/genética , Citrato (si)-Sintase/genética , Ciclo do Ácido Cítrico , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/metabolismo , Metaboloma , Mutagênese Insercional , Fatores de Transcrição/metabolismo
2.
Proc Natl Acad Sci U S A ; 114(31): 8342-8347, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28716935

RESUMO

Viral respiratory infections are usually mild and self-limiting; still they exceptionally result in life-threatening infections in previously healthy children. To investigate a potential genetic cause, we recruited 120 previously healthy children requiring support in intensive care because of a severe illness caused by a respiratory virus. Using exome and transcriptome sequencing, we identified and characterized three rare loss-of-function variants in IFIH1, which encodes an RIG-I-like receptor involved in the sensing of viral RNA. Functional testing of the variants IFIH1 alleles demonstrated that the resulting proteins are unable to induce IFN-ß, are intrinsically less stable than wild-type IFIH1, and lack ATPase activity. In vitro assays showed that IFIH1 effectively restricts replication of human respiratory syncytial virus and rhinoviruses. We conclude that IFIH1 deficiency causes a primary immunodeficiency manifested in extreme susceptibility to common respiratory RNA viruses.


Assuntos
Predisposição Genética para Doença/genética , Síndromes de Imunodeficiência/genética , Helicase IFIH1 Induzida por Interferon/genética , Interferon beta/biossíntese , Vírus Sinciciais Respiratórios/imunologia , Infecções Respiratórias/virologia , Rhinovirus/imunologia , Adenosina Trifosfatases/genética , Pré-Escolar , Cuidados Críticos , Feminino , Variação Genética/genética , Humanos , Síndromes de Imunodeficiência/imunologia , Lactente , Recém-Nascido , Interferon beta/imunologia , Masculino , Estudos Prospectivos , Isoformas de Proteínas/genética , Infecções Respiratórias/imunologia , Replicação Viral/imunologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-28137809

RESUMO

Synthetic peptides derived from the heptad repeat (HR) of fusion (F) proteins can be used as dominant negative inhibitors to inhibit the fusion mechanism of class I viral F proteins. Here, we have performed a stapled-peptide scan across the HR2 domain of the respiratory syncytial virus (RSV) F protein with the aim to identify a minimal domain capable of disrupting the formation of the postfusion six-helix bundle required for viral cell entry. Constraining the peptides with a single staple was not sufficient to inhibit RSV infection. However, the insertion of double staples led to the identification of novel short stapled peptides that display nanomolar potency in HEp-2 cells and are exceptionally robust to proteolytic degradation. By replacing each amino acid of the peptides by an alanine, we found that the substitution of residues 506 to 509, located in a patch of polar contacts between HR2 and HR1, severely affected inhibition. Finally, we show that intranasal delivery of the most potent peptide to BALB/c mice significantly decreased RSV infection in upper and lower respiratory tracts. The discovery of this minimal HR2 sequence as a means for inhibition of RSV infection provides the basis for further medicinal chemistry efforts toward developing RSV fusion antivirals.


Assuntos
Antivirais/farmacologia , Peptídeos/farmacologia , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Proteínas Virais de Fusão/química , Internalização do Vírus/efeitos dos fármacos , Administração Intranasal , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antivirais/síntese química , Sítios de Ligação , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteólise , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Replicação Viral/efeitos dos fármacos
4.
J Virol ; 90(1): 586-90, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26446607

RESUMO

Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-I in vivo, affecting RNA/RIG-I complex formation and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing.


Assuntos
Pareamento Incorreto de Bases , RNA Helicases DEAD-box/metabolismo , Vírus da Influenza A/imunologia , RNA de Cadeia Dupla/genética , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Proteína DEAD-box 58 , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Imunidade Inata , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Cadeia Dupla/química , RNA Viral/química , Receptores Imunológicos
5.
Virology ; 464-465: 330-340, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25113904

RESUMO

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production.


Assuntos
Infecções por Respirovirus/virologia , Vírus Sendai/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo , Membrana Celular/virologia , Humanos , Vírus Sendai/química , Vírus Sendai/genética , Proteínas da Matriz Viral/genética , Vírion/química , Vírion/genética , Vírion/metabolismo , Montagem de Vírus
6.
Virology ; 410(1): 7-16, 2011 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-21075412

RESUMO

Cytoplasmic actins have been found interacting with viral proteins and identified in virus particles. We analyzed by confocal microscopy the cytoplasmic ß- and γ-actin patterns during the course of Sendai virus infections in polarized cells. We observed a spectacular remodeling of the ß-cytoplasmic actin which correlated with productive viral multiplication. Conversely, suppression of M during the course of a productive infection resulted in the decrease of particle production and the absence of ß-actin remodeling. As concomitant suppression of ß- and γ-actins resulted as well in reduction of virus particle production, we propose that Sendai virus specifically induces actin remodeling in order to promote efficient virion production. Beta- and γ-cytoplasmic actin recruitment could substitute for that of the endosomal sorting complex required for transport (ESCRT) mobilized by other enveloped viruses but apparently not used by Sendai virus.


Assuntos
Actinas/metabolismo , Citoplasma/metabolismo , Vírus Sendai/fisiologia , Vírion/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Cães , Regulação Viral da Expressão Gênica/fisiologia , Microscopia Confocal , Isoformas de Proteínas , RNA Interferente Pequeno , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
7.
Virology ; 405(2): 439-47, 2010 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-20633915

RESUMO

Sendai virus (SeV) HN protein is dispensable for virus particle production. HN incorporation into virions strictly depends on a cytoplasmic domain SYWST motif. HNAFYKD, with SYWST replaced with the analogous sequence of measles virus (MeV) H (AFYKD), is not incorporated in virus particles produced by LLCMK2 cells, although it is normally expressed at the plasma membrane. Unlike HNSYWST, HNAFYKD is not internalized to late endosomes, raising the possibility that HN internalization is required for uptake into virus particles. Various mosaic MeV-H containing increasing amounts of the SeV-HN all failed to be taken up in SeV virions. However, when co-expressed with HNAFYKD these MeV-H chimera induced HNAFYKD uptake into virions showing that internalization is not a prerequisite for HN uptake into particles. We propose that HN incorporation in virus particles requires first neutralization by HN of a putative inhibitor of infectious particle formation.


Assuntos
Proteína HN/química , Proteína HN/metabolismo , Vírus Sendai/metabolismo , Vírus Sendai/patogenicidade , Vírion/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Rim/citologia , Rim/virologia , Recombinação Genética , Vírus Sendai/genética
8.
J Virol ; 83(16): 8191-7, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19493999

RESUMO

Members of the Paramyxoviridae such as measles, mumps, and parainfluenza viruses have pleomorphic, enveloped virions that contain negative-sense unsegmented RNA genomes. This is encapsidated by multiple copies of a viral nucleocapsid protein N to form a helical ribonucleoprotein complex (termed the nucleocapsid), which acts as the template for both transcription and replication. Structure analysis of these viruses has proven challenging, owing to disordered regions in important constituent proteins, conformational flexibility in the nucleocapsid and the pleomorphic nature of virus particles. We conducted a low-resolution ultrastructural analysis of Sendai virus, a prototype paramyxovirus, using cryo-electron tomography. Virions are highly variable in size, ranging approximately from 110 to 540 nm in diameter. Envelope glycoproteins are densely packed on the virion surface, while nucleocapsids are clearly resolved in the virion interior. Subtomogram segmentation and filament tracing allowed us to define the path of many nucleocapsids and in some cases to determine the number of putative genomes within a single virus particle. Our findings indicate that these viruses may contain between one and six copies of their genome per virion and that there is no discernible order to nucleocapsid packaging.


Assuntos
Genoma Viral , Vírus Sendai/genética , Vírus Sendai/ultraestrutura , Animais , Embrião de Galinha , Tomografia com Microscopia Eletrônica , Dosagem de Genes , Nucleocapsídeo/química , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Nucleocapsídeo/ultraestrutura , Tamanho da Partícula , Vírus Sendai/química , Vírus Sendai/fisiologia
9.
J Virol ; 81(6): 2861-8, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17192312

RESUMO

Short RNA interference is more and more widely recognized as an effective method to specifically suppress viral functions in eukaryotic cells. Here, we used an experimental system that allows suppression of the Sendai virus (SeV) M protein by using a target sequence, derived from the green fluorescent protein gene, that was introduced in the 3' untranslated region of the M protein mRNA. Silencing of the M protein gene was eventually achieved by a small interfering RNA (siRNA) directed against this target sequence. This siRNA was constitutively expressed in a cell line constructed by transduction with an appropriate lentivirus vector. Suppression of the M protein was sufficient to diminish virus production by 50- to 100-fold. This level of suppression had no apparent effect on viral replication and transcription, supporting the lack of M involvement in SeV transcription or replication control.


Assuntos
Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Viral/biossíntese , Proteínas da Matriz Viral/genética , Vírion/metabolismo , Linhagem Celular Tumoral , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Transdução Genética , Transfecção
10.
Virology ; 344(2): 296-303, 2006 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-16229873

RESUMO

Detergent resistant membranes (DRMs) are the site of assembly for a variety of viruses. Here, we make use of Sendai virus mutant proteins that are not packaged into virus particles to determine the involvement of this assembly for the virus particle production. We found that, in the context of an infection, (1) all the Sendai virus proteins associated in part with DRMs, (2) mutant HN and M proteins not packaged into virus particles were similarly part of this association, (3) after M protein suppression resulting in a significant reduction of virus production, the floatation profile of the other viral proteins was not altered and finally (4) cellular cholesterol depletion did not decrease the virus particle production, although it somehow reduced their virus infectivity. These results led us to conclude that the assembly complex found in DRM fractions does not constitute a direct precursor of virus particle budding.


Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Detergentes/farmacologia , Vírus Sendai/crescimento & desenvolvimento , Montagem de Vírus/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Colesterol/deficiência , Colesterol/metabolismo , Resistência a Medicamentos , Vírus Sendai/metabolismo , Proteínas Virais/metabolismo , Vírion/crescimento & desenvolvimento
11.
J Gen Virol ; 86(Pt 1): 171-180, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15604444

RESUMO

The genomic and antigenomic 3' ends of the Sendai virus replication promoters are bi-partite in nature. They are symmetrically composed of leader or trailer sequences, a gene start (gs) or gene end (ge) site, respectively, and a simple hexameric repeat. Studies of how mRNA synthesis initiates from the first gene start site (gs1) have been hampered by the fact that gs1 is located between two essential elements of the replication promoter. Transcription initiation, then, is separated from the replication initiation site by only 56 nt on the genome, so that transcription and replication may sterically interfere with each other. In order to study the initiation of Sendai virus mRNAs without this possible interference, Sendai virus mini-genomes were prepared having tandem promoters in which replication takes place from the external one, whereas mRNA synthesis occurs from the internal one. Transcription now initiates at position 146 rather than position 56 relative to the genome 3' end. Under these conditions, it was found that the frequency with which mRNA synthesis initiates depends, in an inverse fashion, on the strength of the external replication promoter. It was also found that the sequences essential for replication are not required for basic mRNA synthesis as long as there is an external replication promoter at which viral RNA polymerase can enter the nucleocapsid template. The manner in which transcription and replication initiations influence each other is discussed.


Assuntos
Regiões Promotoras Genéticas/fisiologia , Vírus Sendai/genética , Sítio de Iniciação de Transcrição , Replicação Viral/genética , Regiões 3' não Traduzidas , Sequência de Bases , Proteínas de Fluorescência Verde/biossíntese , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Viral/biossíntese
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