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1.
PLoS Pathog ; 16(8): e1008845, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866210

RESUMO

Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Vírus Vaccinia/fisiologia , Replicação Viral/fisiologia , Células A549 , Animais , Galinhas , Citoplasma/metabolismo , Citoplasma/virologia , DNA Viral/genética , DNA Viral/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Proteínas Repressoras/genética
2.
J Transl Med ; 18(1): 362, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32967693

RESUMO

BACKGROUND: Since the first outbreak of SARS-CoV-2, the clinical characteristics of the Coronavirus Disease 2019 (COVID-19) have been progressively changed. Data reporting a viral intra-host and inter-host evolution favouring the appearance of mild SARS-CoV-2 strains are since being accumulating. To better understand the evolution of SARS-CoV-2 pathogenicity and its adaptation to the host, it is therefore crucial to investigate the genetic and phenotypic characteristics of SARS-CoV-2 strains circulating lately in the epidemic. METHODS: Nasopharyngeal swabs have been analyzed for viral load in the early (March 2020) and late (May 2020) phases of epidemic in Brescia, Italy. Isolation of SARS-CoV-2 from 2 high viral load specimens identified on March 9 (AP66) and on May 8 (GZ69) was performed on Vero E6 cells. Amount of virus released was assessed by quantitative PCR. Genotypic characterization of AP66 and GZ69 was performed by next generation sequencing followed by an in-depth in silico analysis of nucleotide mutations. RESULTS: The SARS-CoV-2 GZ69 strain, isolated in May from an asymptomatic healthcare worker, showed an unprecedented capability of replication in Vero E6 cells in the absence of any evident cytopathic effect. Vero E6 subculturing, up to passage 4, showed that SARS-CoV-2 GZ69 infection was as productive as the one sustained by the cytopathic strain AP66. Whole genome sequencing of the persistently replicating SARS-CoV-2 GZ69 has shown that this strain differs from the early AP66 variant in 9 nucleotide positions (C2939T; C3828T; G21784T; T21846C; T24631C; G28881A; G28882A; G28883C; G29810T) which lead to 6 non-synonymous substitutions spanning on ORF1ab (P892S; S1188L), S (K74N; I95T) and N (R203K, G204R) proteins. CONCLUSIONS: Identification of the peculiar SARS-CoV-2 GZ69 strain in the late Italian epidemic highlights the need to better characterize viral variants circulating among asymptomatic or paucisymptomatic individuals. The current approach could unravel the ways for future studies aimed at analyzing the selection process which favours viral mutations in the human host.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Variação Genética , Pneumonia Viral/virologia , Substituição de Aminoácidos , Animais , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/epidemiologia , Efeito Citopatogênico Viral/genética , Efeito Citopatogênico Viral/fisiologia , Genoma Viral , Humanos , Itália/epidemiologia , Mutação , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Polimorfismo de Nucleotídeo Único , Pesquisa Médica Translacional , Células Vero , Proteínas Virais/genética , Proteínas Virais/fisiologia , Cultura de Vírus/métodos , Replicação Viral/genética , Replicação Viral/fisiologia , Sequenciamento Completo do Genoma
3.
BMJ Open Respir Res ; 7(1)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32913008

RESUMO

We and others propose vimentin as a possible cellular target for the treatment of COVID-19. This innovative idea is so recent that it requires further attention and debate. The significant role played by vimentin in virus-induced infection however is well established: (1) vimentin has been reported as a co-receptor and/or attachment site for SARS-CoV; (2) vimentin is involved in viral replication in cells; (3) vimentin plays a fundamental role in both the viral infection and the consequent explosive immune-inflammatory response and (4) a lower vimentin expression is associated with the inhibition of epithelial to mesenchymal transition and fibrosis. Moreover, the absence of vimentin in mice makes them resistant to lung injury. Since vimentin has a twofold role in the disease, not only being involved in the viral infection but also in the associated life-threatening lung inflammation, the use of vimentin-targeted drugs may offer a synergistic advantage as compared with other treatments not targeting vimentin. Consequently, we speculate here that drugs which decrease the expression of vimentin can be used for the treatment of patients with COVID-19 and advise that several Food and Drug Administration-approved drugs be immediately tested in clinical trials against SARS-CoV-2, thus broadening therapeutic options for this type of viral infection.


Assuntos
Antivirais/farmacologia , Betacoronavirus/fisiologia , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Vimentina/fisiologia , Internalização do Vírus/efeitos dos fármacos , Animais , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação para Baixo , Descoberta de Drogas/métodos , Humanos , Camundongos , Pneumonia/tratamento farmacológico , Pneumonia/imunologia , Pneumonia/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Receptores Virais , Replicação Viral/fisiologia
4.
Nat Commun ; 11(1): 4775, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963221

RESUMO

Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein, thus repressing translation. This mechanism is supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.


Assuntos
Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Sítios Internos de Entrada Ribossomal/fisiologia , Replicação Viral/fisiologia , Regiões 5' não Traduzidas , Linhagem Celular , Infecções por Enterovirus/virologia , Regulação Viral da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , RNA Viral/química , Proteínas Virais/metabolismo
5.
Int J Mol Sci ; 21(16)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824753

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 and its associated pathology, COVID-19, have been of particular concerns these last months due to the worldwide burden they represent. The number of cases requiring intensive care being the critical point in this epidemic, a better understanding of the pathophysiology leading to these severe cases is urgently needed. Tissue lesions can be caused by the pathogen or can be driven by an overwhelmed immune response. Focusing on SARS-CoV-2, we and others have observed that this virus can trigger indeed an immune response that can be dysregulated in severe patients and leading to further injury to multiple organs. The purpose of the review is to bring to light the current knowledge about SARS-CoV-2 virologic and immunologic features. Thus, we address virus biology, life cycle, tropism for many organs and how ultimately it will affect several host biological and physiological functions, notably the immune response. Given that therapeutic avenues are now highly warranted, we also discuss the immunotherapies available to manage the infection and the clinical outcomes.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Fatores Etários , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/terapia , Citocinas/sangue , Humanos , Imunoterapia/métodos , Pulmão/patologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Pneumonia Viral/terapia , RNA Replicase/metabolismo , Proteínas não Estruturais Virais/metabolismo , Tropismo Viral/fisiologia , Montagem de Vírus/fisiologia , Replicação Viral/fisiologia
6.
Nat Commun ; 11(1): 3896, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753727

RESUMO

The outbreak of Zika virus (ZIKV) in 2016 created worldwide health emergency which demand urgent research efforts on understanding the virus biology and developing therapeutic strategies. Here, we present a time-resolved chemical proteomic strategy to track the early-stage entry of ZIKV into host cells. ZIKV was labeled on its surface with a chemical probe, which carries a photocrosslinker to covalently link virus-interacting proteins in living cells on UV exposure at different time points, and a biotin tag for subsequent enrichment and mass spectrometric identification of the receptor or other host proteins critical for virus internalization. We identified Neural Cell Adhesion Molecule (NCAM1) as a potential ZIKV receptor and further validated it through overexpression, knockout, and inhibition of NCAM1 in Vero cells and human glioblastoma cells U-251 MG. Collectively, the strategy can serve as a universal tool to map virus entry pathways and uncover key interacting proteins.


Assuntos
Moléculas de Adesão de Célula Nervosa/metabolismo , Proteômica , Receptores Virais/metabolismo , Internalização do Vírus , Replicação Viral/fisiologia , Zika virus/fisiologia , Animais , Antígeno CD56/genética , Antígeno CD56/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Técnicas de Inativação de Genes , Glioblastoma , Células HEK293 , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Moléculas de Adesão de Célula Nervosa/genética , Células Vero , Proteínas Virais/metabolismo , Ligação Viral , Infecção por Zika virus/virologia
7.
Acta Pharmacol Sin ; 41(9): 1167-1177, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32737471

RESUMO

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus , Medicamentos de Ervas Chinesas , Flavanonas , Flavonoides , Pandemias , Pneumonia Viral , Replicação Viral/efeitos dos fármacos , Administração Oral , Animais , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Ensaios Enzimáticos , Flavanonas/química , Flavanonas/farmacocinética , Flavonoides/química , Flavonoides/farmacocinética , Humanos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Células Vero , Replicação Viral/fisiologia
8.
Vet Microbiol ; 247: 108793, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32768236

RESUMO

Porcine epidemic diarrhea virus (PEDV) belongs to the Alphacoronavirus genus in the Coronaviridae family. Similar to other coronaviruses, PEDV encodes two papain-like proteases. Papain-like protease (PLP)2 has been proposed to play a key role in antagonizing host innate immunity. However, the function of PLP1 remains unclear. In this study, we found that overexpression of PLP1 significantly promoted PEDV replication and inhibited production of interferon-ß. Immunoprecipitation and mass spectrometry were used to identify cellular interaction partners of PLP1. Host cell poly(C) binding protein 2 (PCBP2) was determined to bind and interact with PLP1. Both endogenous and overexpressed PCBP2 co-localized with PLP1 in the cytoplasm. Overexpression of PLP1 upregulated expression of PCBP2. Furthermore, overexpression of PCBP2 promoted PEDV replication. Silencing of endogenous PCBP2 using small interfering RNAs attenuated PEDV replication. Taken together, these data demonstrated that PLP1 negatively regulated the production of type 1 interferon by interacting with PCBP2 and promoted PEDV replication.


Assuntos
Papaína/metabolismo , Vírus da Diarreia Epidêmica Suína/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Interferon beta/genética , Interferon beta/metabolismo , Proteína Proteolipídica de Mielina/metabolismo , Papaína/genética , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/fisiologia , Interferência de RNA , Proteínas de Ligação a RNA , Fator de Necrose Tumoral alfa/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética
9.
Nat Commun ; 11(1): 4070, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792502

RESUMO

Human astroviruses are small non-enveloped viruses with positive-sense single-stranded RNA genomes. Astroviruses cause acute gastroenteritis in children worldwide and have been associated with encephalitis and meningitis in immunocompromised individuals. It is still unknown how astrovirus particles exit infected cells following replication. Through comparative genomic analysis and ribosome profiling we here identify and confirm the expression of a conserved alternative-frame ORF, encoding the protein XP. XP-knockout astroviruses are attenuated and pseudo-revert on passaging. Further investigation into the function of XP revealed plasma and trans Golgi network membrane-associated roles in virus assembly and/or release through a viroporin-like activity. XP-knockout replicons have only a minor replication defect, demonstrating the role of XP at late stages of infection. The discovery of XP advances our knowledge of these important human viruses and opens an additional direction of research into their life cycle and pathogenesis.


Assuntos
Canais Iônicos/metabolismo , Mamastrovirus/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Genômica/métodos , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Canais Iônicos/genética , Mamastrovirus/genética , Microscopia de Fluorescência , Plasmídeos/genética , Ribossomos , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologia
10.
PLoS Negl Trop Dis ; 14(8): e0008469, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32764759

RESUMO

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus, which has rapidly spread around the globe thereby causing millions of infections. CHIKV is an enveloped virus belonging to the Togaviridae family and enters its host cell primarily via clathrin-mediated endocytosis. Upon internalization, the endocytic vesicle containing the virus particle moves through the cell and delivers the virus to early endosomes where membrane fusion is observed. Thereafter, the nucleocapsid dissociates and the viral RNA is translated into proteins. In this study, we examined the importance of the microtubule network during the early steps of infection and dissected the intracellular trafficking behavior of CHIKV particles during cell entry. We observed two distinct CHIKV intracellular trafficking patterns prior to membrane hemifusion. Whereas half of the CHIKV virions remained static during cell entry and fused in the cell periphery, the other half showed fast-directed microtubule-dependent movement prior to delivery to Rab5-positive early endosomes and predominantly fused in the perinuclear region of the cell. Disruption of the microtubule network reduced the number of infected cells. At these conditions, membrane hemifusion activity was not affected yet fusion was restricted to the cell periphery. Furthermore, follow-up experiments revealed that disruption of the microtubule network impairs the delivery of the viral genome to the cell cytosol. We therefore hypothesize that microtubules may direct the particle to a cellular location that is beneficial for establishing infection or aids in nucleocapsid uncoating.


Assuntos
Vírus Chikungunya/fisiologia , Genoma Viral , Microtúbulos/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Humanos , Nocodazol/farmacologia , Moduladores de Tubulina/farmacologia
11.
PLoS Comput Biol ; 16(7): e1007990, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609732

RESUMO

The effective reproduction number, Rt, is a key time-varying prognostic for the growth rate of any infectious disease epidemic. Significant changes in Rt can forewarn about new transmissions within a population or predict the efficacy of interventions. Inferring Rt reliably and in real-time from observed time-series of infected (demographic) data is an important problem in population dynamics. The renewal or branching process model is a popular solution that has been applied to Ebola and Zika virus disease outbreaks, among others, and is currently being used to investigate the ongoing COVID-19 pandemic. This model estimates Rt using a heuristically chosen piecewise function. While this facilitates real-time detection of statistically significant Rt changes, inference is highly sensitive to the function choice. Improperly chosen piecewise models might ignore meaningful changes or over-interpret noise-induced ones, yet produce visually reasonable estimates. No principled piecewise selection scheme exists. We develop a practical yet rigorous scheme using the accumulated prediction error (APE) metric from information theory, which deems the model capable of describing the observed data using the fewest bits as most justified. We derive exact posterior prediction distributions for infected population size and integrate these within an APE framework to obtain an exact and reliable method for identifying the piecewise function best supported by available epidemic data. We find that this choice optimises short-term prediction accuracy and can rapidly detect salient fluctuations in Rt, and hence the infected population growth rate, in real-time over the course of an unfolding epidemic. Moreover, we emphasise the need for formal selection by exposing how common heuristic choices, which seem sensible, can be misleading. Our APE-based method is easily computed and broadly applicable to statistically similar models found in phylogenetics and macroevolution, for example. Our results explore the relationships among estimate precision, forecast reliability and model complexity.


Assuntos
Biologia Computacional/métodos , Infecções por Coronavirus/epidemiologia , Previsões/métodos , Modelos Estatísticos , Pneumonia Viral/epidemiologia , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Surtos de Doenças/prevenção & controle , Humanos , Teoria da Informação , Pandemias , Pneumonia Viral/virologia , Reprodutibilidade dos Testes , Replicação Viral/fisiologia
12.
Nat Commun ; 11(1): 3505, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665593

RESUMO

The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.


Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Linfócitos T CD4-Positivos/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Viral/genética , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Microscopia de Fluorescência , Virologia , Integração Viral/genética , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
13.
PLoS Pathog ; 16(7): e1008644, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678836

RESUMO

The foamy viruses (FV) or spumaviruses are an ancient subfamily of retroviruses that infect a variety of vertebrates. FVs are endemic, but apparently apathogenic, in modern non-human primates. Like other retroviruses, FV replication is inhibited by type-I interferon (IFN). In a previously described screen of IFN-stimulated genes (ISGs), we identified the macaque PHD finger domain protein-11 (PHF11) as an inhibitor of prototype foamy virus (PFV) replication. Here, we show that human and macaque PHF11 inhibit the replication of multiple spumaviruses, but are inactive against several orthoretroviruses. Analysis of other mammalian PHF11 proteins revealed that antiviral activity is host species dependent. Using multiple reporter viruses and cell lines, we determined that PHF11 specifically inhibits a step in the replication cycle that is unique to FVs, namely basal transcription from the FV internal promoter (IP). In so doing, PHF11 prevents expression of the viral transactivator Tas and subsequent activation of the viral LTR promoter. These studies reveal a previously unreported inhibitory mechanism in mammalian cells, that targets a family of ancient viruses and may promote viral latency.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Infecções por Retroviridae/virologia , Spumavirus/fisiologia , Fatores de Transcrição/fisiologia , Latência Viral/fisiologia , Replicação Viral/fisiologia , Animais , Humanos , Macaca
14.
Viruses ; 12(7)2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32630840

RESUMO

Many geometric forms are found in nature, some of them adhering to mathematical laws or amazing aesthetic rules. One of the best-known examples in microbiology is the icosahedral shape of certain viruses with 20 triangular facets and 12 edges. What is less known, however, is that a complementary object displaying 12 faces and 20 edges called a 'dodecahedron' can be produced in huge amounts during certain adenovirus replication cycles. The decahedron was first described more than 50 years ago in the human adenovirus (HAdV3) viral cycle. Later on, the expression of this recombinant scaffold, combined with improvements in cryo-electron microscopy, made it possible to decipher the structural determinants underlying their architecture. Recently, this particle, which mimics viral entry, was used to fish the long elusive adenovirus receptor, desmoglein-2, which serves as a cellular docking for some adenovirus serotypes. This breakthrough enabled the understanding of the physiological role played by the dodecahedral particles, showing that icosahedral and dodecahedral particles live more than a simple platonic story. All these points are developed in this review, and the potential use of the dodecahedron in therapeutic development is discussed.


Assuntos
Adenoviridae/fisiologia , Capsídeo/fisiologia , Infecções por Adenoviridae/patologia , Animais , Proteínas do Capsídeo/fisiologia , Microscopia Crioeletrônica , Humanos , Replicação Viral/fisiologia
15.
Viruses ; 12(7)2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32674326

RESUMO

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 °C, the core body temperature of a chicken, but is able to replicate a 37 °C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins.


Assuntos
Infecções por Coronavirus/prevenção & controle , Vírus da Bronquite Infecciosa/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Linhagem Celular , Embrião de Galinha , Galinhas , Aves Domésticas/virologia , Doenças das Aves Domésticas/virologia , RNA Viral/genética , Temperatura , Vacinas Atenuadas/imunologia , Replicação Viral/genética , Replicação Viral/fisiologia
16.
PLoS Comput Biol ; 16(7): e1008031, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32614817

RESUMO

The 2019-2020 pandemic of atypical pneumonia (COVID-19) caused by the virus SARS-CoV-2 has spread globally and has the potential to infect large numbers of people in every country. Estimating the country-specific basic reproductive ratio is a vital first step in public-health planning. The basic reproductive ratio (R0) is determined by both the nature of pathogen and the network of human contacts through which the disease can spread, which is itself dependent on population age structure and household composition. Here we introduce a transmission model combining age-stratified contact frequencies with age-dependent susceptibility, probability of clinical symptoms, and transmission from asymptomatic (or mild) cases, which we use to estimate the country-specific basic reproductive ratio of COVID-19 for 152 countries. Using early outbreak data from China and a synthetic contact matrix, we estimate an age-stratified transmission structure which can then be extrapolated to 151 other countries for which synthetic contact matrices also exist. This defines a set of country-specific transmission structures from which we can calculate the basic reproductive ratio for each country. Our predicted R0 is critically sensitive to the intensity of transmission from asymptomatic cases; with low asymptomatic transmission the highest values are predicted across Eastern Europe and Japan and the lowest across Africa, Central America and South-Western Asia. This pattern is largely driven by the ratio of children to older adults in each country and the observed propensity of clinical cases in the elderly. If asymptomatic cases have comparable transmission to detected cases, the pattern is reversed. Our results demonstrate the importance of age-specific heterogeneities going beyond contact structure to the spread of COVID-19. These heterogeneities give COVID-19 the capacity to spread particularly quickly in countries with older populations, and that intensive control measures are likely to be necessary to impede its progress in these countries.


Assuntos
Busca de Comunicante/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Previsões/métodos , Modelos Estatísticos , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Fatores Etários , Infecções Assintomáticas/epidemiologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , China/epidemiologia , Biologia Computacional/métodos , Surtos de Doenças/prevenção & controle , Humanos , Pandemias/prevenção & controle , Pandemias/estatística & dados numéricos , Replicação Viral/fisiologia
17.
Proc Natl Acad Sci U S A ; 117(31): 18680-18691, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690711

RESUMO

For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter "crown" complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.


Assuntos
Genoma Viral/genética , RNA Viral/ultraestrutura , Proteínas Virais/ultraestrutura , Replicação Viral/fisiologia , Microscopia Crioeletrônica , Membranas Mitocondriais/ultraestrutura , Modelos Moleculares , Nodaviridae/genética , Nodaviridae/ultraestrutura
18.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32725137

RESUMO

Similar to other RNA viruses, SARS-CoV-2 must (1) enter a target/host cell, (2) reprogram it to ensure its replication, (3) exit the host cell, and (4) repeat this cycle for exponential growth. During the exit step, the virus hijacks the sophisticated machineries that host cells employ to correctly fold, assemble, and transport proteins along the exocytic pathway. Therefore, secretory pathway-mediated assemblage and excretion of infective particles represent appealing targets to reduce the efficacy of virus biogenesis, if not to block it completely. Here, we analyze and discuss the contribution of the molecular machines operating in the early secretory pathway in the biogenesis of SARS-CoV-2 and their relevance for potential antiviral targeting. The fact that these molecular machines are conserved throughout evolution, together with the redundancy and tissue specificity of their components, provides opportunities in the search for unique proteins essential for SARS-CoV-2 biology that could also be targeted with therapeutic objectives. Finally, we provide an overview of recent evidence implicating proteins of the early secretory pathway as potential antiviral targets with effective therapeutic applications.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Via Secretória/fisiologia , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Via Secretória/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
19.
Rev Med Interne ; 41(6): 375-389, 2020 Jun.
Artigo em Francês | MEDLINE | ID: covidwho-578061

RESUMO

SARS-CoV-2 infection, named COVID-19, can lead to a dysregulated immune response and abnormal coagulation responsible for a viral sepsis. In this review, we specify physiopathological mechanisms of each phase of COVID-19 - viral, immune and pro-thrombotic - notably because they involve different treatment. Finally, we specify the physiopathological mechanisms of organ injury.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Animais , Betacoronavirus/genética , Betacoronavirus/imunologia , Betacoronavirus/ultraestrutura , Infecções por Coronavirus/complicações , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Citocinas/metabolismo , Humanos , Imunização , Imunomodulação , Especificidade de Órgãos , Pneumonia Viral/complicações , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Trombose/prevenção & controle , Trombose/virologia , Tropismo Viral , Internalização do Vírus , Replicação Viral/fisiologia , Zoonoses/virologia
20.
Rev Med Interne ; 41(6): 375-389, 2020 Jun.
Artigo em Francês | MEDLINE | ID: covidwho-601583

RESUMO

SARS-CoV-2 infection, named COVID-19, can lead to a dysregulated immune response and abnormal coagulation responsible for a viral sepsis. In this review, we specify physiopathological mechanisms of each phase of COVID-19 - viral, immune and pro-thrombotic - notably because they involve different treatment. Finally, we specify the physiopathological mechanisms of organ injury.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Animais , Betacoronavirus/genética , Betacoronavirus/imunologia , Betacoronavirus/ultraestrutura , Infecções por Coronavirus/complicações , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Citocinas/metabolismo , Humanos , Imunização , Imunomodulação , Especificidade de Órgãos , Pneumonia Viral/complicações , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Trombose/prevenção & controle , Trombose/virologia , Tropismo Viral , Internalização do Vírus , Replicação Viral/fisiologia , Zoonoses/virologia
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