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1.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803568

RESUMO

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein-cargo and protein-protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.


Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Vírion/metabolismo , Bromovirus/ultraestrutura , Modelos Moleculares , Tamanho da Partícula , RNA de Transferência/metabolismo , Temperatura , Vírion/ultraestrutura
2.
Nat Commun ; 12(1): 2149, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846319

RESUMO

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, ß1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct ß1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and ß1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and ß1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for ß1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and ß1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.


Assuntos
Clatrina/metabolismo , Interações Hospedeiro-Patógeno , Integrina beta1/metabolismo , Reoviridae/fisiologia , Animais , Sítios de Ligação , Capsídeo/metabolismo , Cátions , Linhagem Celular , Membrana Celular/metabolismo , Endocitose , Cinética , Camundongos , Ácido N-Acetilneuramínico/metabolismo , Mutação Puntual/genética , Ligação Proteica , Termodinâmica , Proteínas Virais/metabolismo , Vírion/metabolismo
3.
Arch Virol ; 166(5): 1371-1383, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33715038

RESUMO

Herpesviruses are capable of infecting not only neurons, where they establish latent infection, but also astrocytes. Since astrocytes are important for the functioning of the central nervous system (CNS), their infection may lead to serious neurological disorders. Thus, in the present study we investigated the ability of human herpesvirus type 2 (HHV-2) to infect primary murine astrocytes in vitro and the effect of infection on their mitochondrial network and actin cytoskeleton. In immunofluorescence assays, antibodies against HHV-2 antigens and glial fibrillary acidic protein (GFAP) were used to confirm that the infected cells are indeed astrocytes. Real-time PCR analysis showed a high level of HHV-2 replication in astrocytes, particularly at 168 h postinfection, confirming that a productive infection had occurred. Analysis of mitochondrial morphology showed that, starting from the first stage of infection, HHV-2 caused fragmentation of the mitochondrial network and formation of punctate and tubular structures that colocalized with virus particles. Furthermore, during the late stages of infection, the infection affected the actin cytoskeleton and induced formation of actin-based cellular projections, which were probably associated with enhanced intracellular spread of the virus. These results suggest that the observed changes in the mitochondrial network and actin cytoskeleton in productively infected astrocytes are required for effective replication and viral spread in a primary culture of astrocytes. Moreover, we speculate that, in response to injury such as HHV-2 infection, murine astrocytes cultured in vitro undergo transformation, defined in vivo as reactive astrocytosis.


Assuntos
Citoesqueleto de Actina/patologia , Astrócitos/virologia , Herpesvirus Humano 2/fisiologia , Mitocôndrias/patologia , Citoesqueleto de Actina/metabolismo , Animais , Astrócitos/patologia , Células Cultivadas , Gliose , Cinética , Camundongos , Mitocôndrias/metabolismo , Vírion/metabolismo , Replicação Viral
4.
Adv Colloid Interface Sci ; 290: 102400, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33713994

RESUMO

We review concepts involved in describing the chemodynamic features of nanoparticles and apply the framework to gain physicochemical insights into interactions between SARS-CoV-2 virions and airborne particulate matter (PM). Our analysis is highly pertinent given that the World Health Organisation acknowledges that SARS-CoV-2 may be transmitted by respiratory droplets, and the US Center for Disease Control and Prevention recognises that airborne transmission of SARS-CoV-2 can occur. In our theoretical treatment, the virion is assimilated to a core-shell nanoparticle, and contributions of various interaction energies to the virion-PM association (electrostatic, hydrophobic, London-van der Waals, etc.) are generically included. We review the limited available literature on the physicochemical features of the SARS-CoV-2 virion and identify knowledge gaps. Despite the lack of quantitative data, our conceptual framework qualitatively predicts that virion-PM entities are largely able to maintain equilibrium on the timescale of their diffusion towards the host cell surface. Comparison of the relevant mass transport coefficients reveals that virion biointernalization demand by alveolar host cells may be greater than the diffusive supply. Under such conditions both the free and PM-sorbed virions may contribute to the transmitted dose. This result points to the potential for PM to serve as a shuttle for delivery of virions to host cell targets. Thus, our critical review reveals that the chemodynamics of virion-PM interactions may play a crucial role in the transmission of COVID-19, and provides a sound basis for explaining reported correlations between episodes of air pollution and outbreaks of COVID-19.


Assuntos
/transmissão , Células Epiteliais/virologia , Material Particulado/química , Vírion/química , Aerossóis , Fenômenos Biomecânicos , Difusão , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Químicos , Nanopartículas/química , Alvéolos Pulmonares/virologia , /patogenicidade , Eletricidade Estática , Vírion/metabolismo , Vírion/patogenicidade , Internalização do Vírus , Água/química
5.
Nat Commun ; 12(1): 1658, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712578

RESUMO

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.


Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Vírus da Hepatite B/fisiologia , Replicação Viral/fisiologia , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/genética , Ritmo Circadiano/genética , DNA Circular , DNA Viral/metabolismo , Regulação da Expressão Gênica , Genoma Viral , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Hepatócitos/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Fígado/metabolismo , Camundongos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Regiões Promotoras Genéticas , Simportadores/metabolismo , Transcriptoma , Vírion/metabolismo , Internalização do Vírus
6.
mBio ; 12(2)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727347

RESUMO

An emerging class of cellular inhibitory proteins has been identified that targets viral glycoproteins. These include the membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases that, among other functions, downregulate cell surface proteins involved in adaptive immunity. The RING-CH domain of MARCH proteins is thought to function by catalyzing the ubiquitination of the cytoplasmic tails (CTs) of target proteins, leading to their degradation. MARCH proteins have recently been reported to target retroviral envelope glycoproteins (Env) and vesicular stomatitis virus G glycoprotein (VSV-G). However, the mechanism of antiviral activity remains poorly defined. Here we show that MARCH8 antagonizes the full-length forms of HIV-1 Env, VSV-G, Ebola virus glycoprotein (EboV-GP), and the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), thereby impairing the infectivity of virions pseudotyped with these viral glycoproteins. This MARCH8-mediated targeting of viral glycoproteins requires the E3 ubiquitin ligase activity of the RING-CH domain. We observe that MARCH8 protein antagonism of VSV-G is CT dependent. In contrast, MARCH8-mediated targeting of HIV-1 Env, EboV-GP, and SARS-CoV-2 S protein by MARCH8 does not require the CT, suggesting a novel mechanism of MARCH-mediated antagonism of these viral glycoproteins. Confocal microscopy data demonstrate that MARCH8 traps the viral glycoproteins in an intracellular compartment. We observe that the endogenous expression of MARCH8 in several relevant human cell types is rapidly inducible by type I interferon. These results help to inform the mechanism by which MARCH proteins exert their antiviral activity and provide insights into the role of cellular inhibitory factors in antagonizing the biogenesis, trafficking, and virion incorporation of viral glycoproteins.IMPORTANCE Viral envelope glycoproteins are an important structural component on the surfaces of enveloped viruses that direct virus binding and entry and also serve as targets for the host adaptive immune response. In this study, we investigate the mechanism of action of the MARCH family of cellular proteins that disrupt the trafficking and virion incorporation of viral glycoproteins across several virus families. This research provides novel insights into how host cell factors antagonize viral replication, perhaps opening new avenues for therapeutic intervention in the replication of a diverse group of highly pathogenic enveloped viruses.


Assuntos
Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Interferons/farmacologia , Espaço Intracelular/metabolismo , Proteínas de Membrana/genética , Mutação , Vírus de RNA/classificação , Vírus de RNA/metabolismo , Especificidade da Espécie , Ubiquitina-Proteína Ligases/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vírion/metabolismo , Replicação Viral
7.
Methods Mol Biol ; 2244: 213-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555589

RESUMO

Human cytomegalovirus (HCMV) is a large double-stranded DNA virus and member of the ß-herpesvirus family. HCMV is ubiquitous in the human population and causes lifelong infections. HCMV infection is associated with high morbidity and mortality in immunocompromised individuals and the virus is a major cause of virus-mediated congenital disease. There have been a number of HCMV entry receptors identified that use one of two viral receptor binding complexes, including the gH/gL/gO complex and the pentamer made up of gH/gL/UL128/UL130/UL131a. Cytomegaloviruses (CMVs) are typically host-restricted requiring the use of species-specific modeling and culture conditions. We use rat CMV (RCMV) to study CMV-accelerated vascular disease and chronic allograft rejection. RCMV encodes homologous versions of the entry complex proteins but their incorporation and copy number per virion are still unknown. In this methods article, we describe a novel approach of HiBiT tagging viral proteins in order to detect and quantify protein incorporation into particles. This method is independent of protein-specific antibodies and can be standardized using a commercially available HiBiT protein standard. Using bacterial artificial chromosome (BAC) recombineering, we have constructed two individual viruses containing a HiBiT tag fused to the C'-terminus of either the UL128 homolog (R129) or the UL130 homolog (R131). Viruses containing these mutations were rescued, purified and analyzed. Our data demonstrate that R129 and R131 are both incorporated into RCMV virions at equimolar ratios relative to genome copy number, supporting this antibody-free approach for quantifying viral protein incorporation and its application toward the identification of domains required for incorporation.


Assuntos
Medições Luminescentes/métodos , Proteínas Luminescentes/síntese química , Animais , Cromossomos Artificiais Bacterianos/genética , Citomegalovirus/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/genética , Ligação Proteica , Ratos , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Vírion/metabolismo , Internalização do Vírus
8.
Methods Mol Biol ; 2244: 265-289, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33555592

RESUMO

The generation and release of mature virions from human cytomegalovirus (HCMV) infected cells is a multistep process, involving a profound reorganization of cellular structures and various stages of virus particle morphogenesis in different cellular compartments. Although the general steps of HCMV morphogenesis are known, it has become clear that the detailed molecular mechanisms are complex and dependent on various viral factors and cellular pathways. The lack of a full understanding of HCMV virion morphogenesis emphasizes the need of imaging techniques to visualize the different stages of virion assembly, such as electron microscopy. Here, we describe various electron microscopy techniques and the methodology of high-pressure freezing and freeze substitution for sample preparation to visualize HCMV morphogenesis. These methods are used in our laboratory in combination with a thorough quantification to characterize phenotypic alterations and to identify the function of viral and cellular proteins for the various morphogenesis stages.


Assuntos
Infecções por Citomegalovirus/diagnóstico por imagem , Citomegalovirus/crescimento & desenvolvimento , Microscopia Eletrônica/métodos , Linhagem Celular , Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Citoplasma/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Morfogênese/fisiologia , Transporte Proteico/fisiologia , Proteínas Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus/fisiologia , Replicação Viral/fisiologia
9.
Virology ; 556: 9-22, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33524849

RESUMO

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.


Assuntos
Infecções por Coronavirus/virologia , Retículo Endoplasmático/metabolismo , Vírus da Hepatite Murina/fisiologia , /metabolismo , Animais , Linhagem Celular , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Lisossomos/virologia , Camundongos , Proteínas Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus , Replicação Viral
10.
Viruses ; 13(2)2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525505

RESUMO

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.


Assuntos
Antivirais/farmacologia , Quadruplex G/efeitos dos fármacos , Herpesvirus Humano 1/efeitos dos fármacos , Porfirinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , DNA Viral/química , DNA Viral/efeitos dos fármacos , Herpesvirus Humano 1/fisiologia , Ligantes , Estrutura Molecular , Porfirinas/química , Células Vero , Vírion/efeitos dos fármacos , Vírion/metabolismo
11.
Virology ; 556: 1-8, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33515858

RESUMO

Porcine deltacoronavirus (PDCoV) is one of the emerged coronaviruses posing a significant threat to the swine industry. Previous work showed the presence of a viral accessory protein NS6 in PDCoV-infected cells. In this study, we detected the expression of the NS6 protein in small intestinal tissues of PDCoV-infected piglets. In addition, SDS-PAGE and Western blot analysis of sucrose gradient-purified virions showed the presence of a 13-kDa NS6 protein. Further evidences of the presence of NS6 in the PDCoV virions were obtained by immunogold staining of purified virions with anti-NS6 antiserum, and by immunoprecipitation of NS6 from purified virions. Finally, the anti-NS6 antibody was not able to neutralize PDCoV in cultured cells. These data establish for the first time that the accessory protein NS6 is expressed during infection in vivo and incorporated into PDCoV virions.


Assuntos
Infecções por Coronavirus/veterinária , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/metabolismo , Vírion/metabolismo , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/virologia , Camundongos , Coelhos , Suínos , Doenças dos Suínos/metabolismo , Proteínas não Estruturais Virais/imunologia
12.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33408170

RESUMO

Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Although cholesterol has been associated with the binding and entry of various enveloped viruses into cells, cholesterol's exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MßCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (VSV)-pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes.IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here, we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytium formation and virus-cell membrane fusion during viral entry. Furthermore, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.


Assuntos
Colesterol/metabolismo , Fusão de Membrana/fisiologia , Vírus Nipah/fisiologia , Colesterol/deficiência , Células Gigantes/metabolismo , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Vírus Nipah/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/metabolismo , Internalização do Vírus
13.
Nat Commun ; 12(1): 98, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397935

RESUMO

Glucose metabolism and innate immunity evolved side-by-side. It is unclear if and how the two systems interact with each other during hepatitis B virus (HBV) infections and, if so, which mechanisms are involved. Here, we report that HBV activates glycolysis to impede retinoic acid-inducible gene I (RIG-I)-induced interferon production. We demonstrate that HBV sequesters MAVS from RIG-I by forming a ternary complex including hexokinase (HK). Using a series of pharmacological and genetic approaches, we provide in vitro and in vivo evidence indicating that HBV suppresses RLR signaling via lactate dehydrogenase-A-dependent lactate production. Lactate directly binds MAVS preventing its aggregation and mitochondrial localization during HBV infection. Therefore, we show that HK2 and glycolysis-derived lactate have important functions in the immune escape of HBV and that energy metabolism regulates innate immunity during HBV infection.


Assuntos
Vírus da Hepatite B/fisiologia , Imunidade Inata , Metaboloma , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anaerobiose , Animais , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Glucose/metabolismo , Glicólise , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Humanos , Evasão da Resposta Imune , Interferons/metabolismo , Ácido Láctico/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transdução de Sinais , Vírion/metabolismo
14.
Methods Mol Biol ; 2225: 163-177, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33108662

RESUMO

Viruses engineered to express fluorescent proteins can be used with live-cell imaging techniques to monitor the progression of infection in real time. Here we describe a set of methods to track infection spreading from one cell population to another as well as to visualize transfer of virions between cells. This approach is extended to multiplexing with physiological readouts of cell death, which can be correlated with single-cell resolution to viral infection.


Assuntos
Rastreamento de Células/métodos , Engenharia Genética/métodos , Myxoma virus/genética , Imagem Óptica/métodos , Proteínas Virais/genética , Vírion/genética , Células A549 , Apoptose/genética , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Myxoma virus/crescimento & desenvolvimento , Myxoma virus/metabolismo , Imagem Óptica/estatística & dados numéricos , Proteínas Virais/metabolismo , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Replicação Viral/genética
15.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33328311

RESUMO

Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome.IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.


Assuntos
Genoma Viral/genética , Polimorfismo Genético , Infecção por Zika virus/virologia , Zika virus/genética , Aedes/virologia , Animais , Linhagem Celular , Variações do Número de Cópias de DNA , Tamanho do Genoma , Genótipo , Humanos , Mosquitos Vetores/virologia , Temperatura , Vírion/metabolismo , Replicação Viral , Zika virus/crescimento & desenvolvimento , Infecção por Zika virus/transmissão
16.
Sci Rep ; 10(1): 21877, 2020 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-33318562

RESUMO

SARS-CoV-2 virus is the causative agent of COVID-19. Here we demonstrate that non-infectious SARS-CoV-2 virus like particles (VLPs) can be assembled by co-expressing the viral proteins S, M and E in mammalian cells. The assembled SARS-CoV-2 VLPs possess S protein spikes on particle exterior, making them ideal for vaccine development. The particles range in shape from spherical to elongated with a characteristic size of 129 ± 32 nm. We further show that SARS-CoV-2 VLPs dried in ambient conditions can retain their structural integrity upon repeated scans with Atomic Force Microscopy up to a peak force of 1 nN.


Assuntos
/virologia , Vírion/metabolismo , Montagem de Vírus , Células HEK293 , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas da Matriz Viral/metabolismo
17.
Cell Rep ; 33(12): 108528, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33326798

RESUMO

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.


Assuntos
/metabolismo , Antivirais/farmacologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Microcorpos/metabolismo , /efeitos dos fármacos , Sequência de Aminoácidos , Animais , /virologia , Modelos Animais de Doenças , Dissulfetos/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Domínios Proteicos , Multimerização Proteica , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacos
18.
BMC Pulm Med ; 20(1): 301, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198751

RESUMO

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly reached pandemic proportions. Given that the main target of SARS-CoV-2 are lungs leading to severe pneumonia with hyperactivation of the inflammatory cascade, we conducted a prospective study to assess alveolar inflammatory status in patients with moderate to severe COVID-19. METHODS: Diagnostic bronchoalveolar lavage (BAL) was performed in 33 adult patients with SARS-CoV-2 infection by real-time PCR on nasopharyngeal swab admitted to the Intensive care unit (ICU) (n = 28) and to the Intermediate Medicine Ward (IMW) (n = 5). We analyze the differential cell count, ultrastructure of cells and Interleukin (IL)6, 8 and 10 levels. RESULTS: ICU patients showed a marked increase in neutrophils (1.24 × 105 ml- 1, 0.85-2.07), lower lymphocyte (0.97 × 105 ml- 1, 0.024-0.34) and macrophages fractions (0.43 × 105 ml- 1, 0.34-1.62) compared to IMW patients (0.095 × 105 ml- 1, 0.05-0.73; 0.47 × 105 ml- 1, 0.28-1.01 and 2.14 × 105 ml- 1, 1.17-3.01, respectively) (p < 0.01). Study of ICU patients BAL by electron transmission microscopy showed viral particles inside mononuclear cells confirmed by immunostaining with anti-viral capsid and spike antibodies. IL6 and IL8 were significantly higher in ICU patients than in IMW (IL6 p < 0.01, IL8 p < 0.0001), and also in patients who did not survive (IL6 p < 0.05, IL8 p = 0.05 vs. survivors). IL10 did not show a significant variation between groups. Dividing patients by treatment received, lower BAL concentrations of IL6 were found in patients treated with steroids as compared to those treated with tocilizumab (p < 0.1) or antivirals (p < 0.05). CONCLUSIONS: Alveolitis, associated with COVID-19, is mainly sustained by innate effectors which showed features of extensive activation. The burden of pro-inflammatory cytokines IL6 and IL8 in the broncho-alveolar environment is associated with clinical outcome.


Assuntos
Líquido da Lavagem Broncoalveolar/imunologia , Infecções por Coronavirus/imunologia , Inflamação/imunologia , Interleucina-6/imunologia , Interleucina-8/imunologia , Leucócitos/imunologia , Pulmão/imunologia , Macrófagos Alveolares/imunologia , Pneumonia Viral/imunologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Corticosteroides/uso terapêutico , Idoso , Alanina/análogos & derivados , Alanina/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus , Lavagem Broncoalveolar , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/virologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/terapia , Combinação de Medicamentos , Feminino , Humanos , Hidroxicloroquina/uso terapêutico , Unidades de Terapia Intensiva , Interleucina-10/imunologia , Itália , Leucócitos Mononucleares/virologia , Lopinavir/uso terapêutico , Pulmão/citologia , Pulmão/virologia , Linfócitos/imunologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Neutrófilos/imunologia , Pandemias , Pneumonia Viral/terapia , Prognóstico , Estudos Prospectivos , Respiração Artificial/métodos , Ritonavir/uso terapêutico , Glicoproteína da Espícula de Coronavírus/metabolismo , Taxa de Sobrevida , Vírion/metabolismo , Vírion/ultraestrutura
19.
Nat Commun ; 11(1): 5885, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208793

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic ß-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Replicação Viral , Células A549 , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Vesículas Citoplasmáticas/virologia , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/virologia , Humanos , Pandemias , Pneumonia Viral/virologia , RNA Viral/química , RNA Viral/metabolismo , Células Vero , Vírion/química , Vírion/metabolismo , Montagem de Vírus
20.
Viruses ; 12(10)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003350

RESUMO

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2' cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Vírus da Bronquite Infecciosa/efeitos dos fármacos , Vírus da Bronquite Infecciosa/fisiologia , Tripsina/uso terapêutico , Tropismo Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Gammacoronavirus/efeitos dos fármacos , Vírus da Bronquite Infecciosa/metabolismo , Cinética , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Proteínas do Envelope Viral/metabolismo , Vírion/efeitos dos fármacos , Vírion/metabolismo , Replicação Viral/efeitos dos fármacos
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