RESUMO
Rift Valley fever virus (RVFV) is a pathogen transmitted to humans and livestock via mosquito bites. This virus, which was discovered in Kenya in 1930, is considered by the World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) to be associated with a high risk of causing large-scale epidemics. However, means dedicated to fighting RVFV have been limited, and despite recent research efforts, the virus remains poorly understood at both the molecular and cellular levels as well as at a broader scale of research in the field and in animal and human populations. In this introductory chapter of a methods book, we aim to provide readers with a concise overview of RVFV, from its ecology and transmission to the structural and genomic organization of virions and its life cycle in host cells.
Assuntos
Febre do Vale de Rift , Vírus da Febre do Vale do Rift , Vírus da Febre do Vale do Rift/genética , Febre do Vale de Rift/transmissão , Febre do Vale de Rift/virologia , Animais , Humanos , Genoma ViralRESUMO
Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that represents a significant threat to both human and veterinary public health. Since its discovery in the Great Rift Valley of Kenya in the 1930s, the virus has spread across Africa and beyond, now posing a risk of introduction into Southern Europe and Asia. Despite recent progresses, early RVFV-host cell interactions remain largely uncharacterized. In this method chapter, we delineate the procedure for labeling RVFV particles with fluorescent organic dyes. This approach makes it feasible to visualize single viral particles in both fixed and living cells and study RVFV entry into host cells. We provide additional examples with two viruses closely related to RVFV, namely, Toscana virus and Uukuniemi virus. Furthermore, we illustrate how to utilize fluorescent viral particles to examine and quantify each step of the cell entry program of RVFV, which includes state-of-the-art fluorescence-based detection techniques such as fluorescence microscopy, flow cytometry, and fluorimetry.
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Corantes Fluorescentes , Microscopia de Fluorescência , Vírus da Febre do Vale do Rift , Vírion , Vírus da Febre do Vale do Rift/isolamento & purificação , Humanos , Vírion/isolamento & purificação , Animais , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Citometria de Fluxo/métodos , Internalização do Vírus , Febre do Vale de Rift/virologia , Febre do Vale de Rift/diagnóstico , Coloração e Rotulagem/métodos , Linhagem CelularRESUMO
Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.
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Orthobunyavirus , Glucosilceramidas , Ligação Viral , Lipidômica , Espectrometria de MassasRESUMO
Bunyavirales constitute the largest order of enveloped RNA viruses, many members of which cause severe diseases in humans and domestic animals. In recent decades, innovative fluorescence-based methods have paved the way to visualize and track single fluorescent bunyaviral particles in fixed and live cells. This technological breakthrough has enabled imaging of the early stages of infection and the quantification of every step in the bunyavirus cell entry process. Here, we describe the latest procedures for rendering bunyaviral particles fluorescent and discuss the advantages and disadvantages of each approach in light of the most recent advances in fluorescence detection and monitoring of bunyavirus entry. In this mini-review, we also illustrate how fluorescent viral particles are a powerful tool for deciphering the cellular entry process of bunyaviruses, the vast majority of which have not yet been analyzed.
Assuntos
Orthobunyavirus , Vírus de RNA , Animais , Humanos , Fluorescência , Internalização do VírusRESUMO
For successful infection of host cells and virion production, enveloped viruses, including Zika virus (ZIKV), extensively rely on cellular lipids. However, how virus protein-lipid interactions contribute to the viral life cycle remains unclear. Here, we employ a chemo-proteomics approach with a bifunctional cholesterol probe and show that cholesterol is closely associated with the ZIKV structural protein prM. Bioinformatic analyses, reverse genetics alongside with photoaffinity labeling assays, and atomistic molecular dynamics simulations identified two functional cholesterol binding motifs within the prM transmembrane domain. Loss of prM-cholesterol association has a bipartite effect reducing ZIKV entry and leading to assembly defects. We propose a model in which membrane-resident M facilitates cholesterol-supported lipid exchange during endosomal entry and, together with cholesterol, creates a platform promoting virion assembly. In summary, we identify a bifunctional role of prM in the ZIKV life cycle by mediating viral entry and virus assembly in a cholesterol-dependent manner.
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Infecção por Zika virus , Zika virus , Humanos , Zika virus/metabolismo , Internalização do Vírus , Replicação Viral , Proteínas Virais/metabolismo , LipídeosRESUMO
Amyloidoses are an array of diseases associated with the aggregation of proteins into fibrils. While it was previously thought that amyloid fibril-forming proteins are exclusively host-cell encoded, recent studies have revealed that pathogenic viruses can form amyloid-like fibrils too. Intriguingly, viral amyloids are often composed of virulence factors, known for their contribution to cell death and disease progression. In this review, we survey the literature about viral proteins capable of forming amyloid-like fibrils. The molecular and cellular mechanisms underlying the formation of viral amyloid-like aggregates are explored. In addition, we discuss the functional implications for viral amplification and the complex interplay between viral amyloids, biological functions, virulence, and virus-induced pathologies.
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Amiloide , Proteínas Amiloidogênicas , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Fatores de Virulência , AntiviraisRESUMO
Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.
Assuntos
Vírus da Febre do Flebótomo Napolitano , Humanos , Endocitose , Endossomos/metabolismo , Vacúolos , Internalização do Vírus , Concentração de Íons de HidrogênioRESUMO
With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30 to 40 min. The virus entered Rab5a-positive (Rab5a+) early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15 to 25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. IMPORTANCE Orthobunyaviruses (OBVs), which include La Crosse, Oropouche, and Schmallenberg viruses, represent a growing threat to humans and domestic animals worldwide. Ideally, preventing OBV spread requires approaches that target early stages of infection, i.e., virus entry. However, little is known about the molecular and cellular mechanisms by which OBVs enter and infect host cells. Here, we developed accurate, sensitive tools and assays to investigate the penetration process of GERV. Our data emphasize the central role of late endosomal maturation in GERV entry, providing a comprehensive overview of the early stages of an OBV infection. Our study also brings a complete toolbox of innovative methods to study each step of the OBV entry program in fixed and living cells, from virus binding and endocytosis to fusion and penetration. The information gained herein lays the foundation for the development of antiviral strategies aiming to block OBV entry.
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Endossomos , Orthobunyavirus , Internalização do Vírus , Animais , Microscopia Crioeletrônica , Endossomos/virologia , Mamíferos , Orthobunyavirus/fisiologiaRESUMO
The most severe forms of coronavirus disease 2019 (COVID-19) are often associated with the presence of syncytia in the lungs resulting from cell-cell fusion mediated by the SARS-CoV-2 spike protein. In this issue, Rajah and colleagues show that the SARS-CoV-2 alpha, beta, and delta variants promote enhanced syncytia formation as compared to the original strain.
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COVID-19 , SARS-CoV-2 , Humanos , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
SARS-CoV-2 is a newly emerged coronavirus that caused the global COVID-19 outbreak in early 2020. COVID-19 is primarily associated with lung injury, but many other clinical symptoms such as loss of smell and taste demonstrated broad tissue tropism of the virus. Early SARS-CoV-2-host cell interactions and entry mechanisms remain poorly understood. Investigating SARS-CoV-2 infection in tissue culture, we found that the protease TMPRSS2 determines the entry pathway used by the virus. In the presence of TMPRSS2, the proteolytic process of SARS-CoV-2 was completed at the plasma membrane, and the virus rapidly entered the cells within 10 min in a pH-independent manner. When target cells lacked TMPRSS2 expression, the virus was endocytosed and sorted into endolysosomes, from which SARS-CoV-2 entered the cytosol via acid-activated cathepsin L protease 40-60 min post-infection. Overexpression of TMPRSS2 in non-TMPRSS2 expressing cells abolished the dependence of infection on the cathepsin L pathway and restored sensitivity to the TMPRSS2 inhibitors. Together, our results indicate that SARS-CoV-2 infects cells through distinct, mutually exclusive entry routes and highlight the importance of TMPRSS2 for SARS-CoV-2 sorting into either pathway.
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COVID-19/metabolismo , Catepsina L/metabolismo , SARS-CoV-2/fisiologia , Serina Endopeptidases/metabolismo , Animais , COVID-19/genética , Células CACO-2 , Chlorocebus aethiops , Endocitose , Interações entre Hospedeiro e Microrganismos , Humanos , Concentração de Íons de Hidrogênio , Proteólise , Serina Endopeptidases/genética , Transdução de Sinais , Células Vero , Internalização do VírusRESUMO
With over 80 members worldwide, Orthobunyavirus is the largest genus in the Peribunyaviridae family. Orthobunyaviruses (OBVs) are arthropod-borne viruses that are structurally simple, with a trisegmented, negative-sense RNA genome and only four structural proteins. OBVs are potential agents of emerging and re-emerging diseases and overall represent a global threat to both public and veterinary health. The focus of this review is on the very first steps of OBV infection in mammalian hosts, from virus binding to penetration and release of the viral genome into the cytosol. Here, we address the most current knowledge and advances regarding OBV receptors, endocytosis, and fusion.
Assuntos
Infecções por Bunyaviridae/virologia , Orthobunyavirus/fisiologia , Ligação Viral , Internalização do Vírus , Animais , Transporte Biológico , Membrana Celular/metabolismo , Genoma Viral , Interações Hospedeiro-Patógeno , Humanos , Especificidade da Espécie , Tropismo Viral , VírionRESUMO
Rift Valley Fever Virus (RVFV) is an emerging zoonotic pathogen transmitted to humans and livestock through mosquito bites, which was first isolated in Kenya in 1930. The virus is classified by the WHO among the pathogens for which there is an urgent need to develop research, diagnostics, and therapies. However, the efforts developed to control the virus remain limited, and the virus is not well characterized. In this article, we will introduce RVFV and then focus on its virulence factor, the nonstructural protein NSs. We will mainly discuss the ability of this viral protein to form amyloid-like fibrils and its implication in the neurotoxicity associated with RVFV infection.
TITLE: Le virus de la fièvre de la vallée du Rift et son étonnante protéine NSs. ABSTRACT: Le virus de la fièvre de la vallée du Rift (VFVR) est un agent pathogène transmis à l'homme et au bétail par la piqûre de moustiques. Ce virus, découvert au Kenya en 1930, est considéré par l'Organisation mondiale de la santé comme présentant un risque important de provoquer de vastes épidémies. Les moyens dédiés à la lutte contre le VFVR restent toutefois particulièrement limités et le virus est mal connu. Dans cette Synthèse, nous nous attacherons à présenter ce virus avant de nous intéresser plus spécifiquement à son facteur de virulence, la protéine NSs. Nous discuterons la capacité de cette protéine virale à former des fibrilles de type amyloïde et son implication dans la neurotoxicité du virus chez les animaux infectés.
Assuntos
Vírus da Febre do Vale do Rift , Animais , Humanos , Vírus da Febre do Vale do Rift/genética , Proteínas não Estruturais Virais/genética , Fatores de VirulênciaRESUMO
Phenuiviridae is a large family of arthropod-borne viruses with over 100 species worldwide. Several cause severe diseases in both humans and livestock. Global warming and the apparent geographical expansion of arthropod vectors are good reasons to seriously consider these viruses potential agents of emerging diseases. With an increasing frequency and number of epidemics, some phenuiviruses represent a global threat to public and veterinary health. This review focuses on the early stage of phenuivirus infection in mammalian host cells. We address current knowledge on each step of the cell entry process, from virus binding to penetration into the cytosol. Virus receptors, endocytosis, and fusion mechanisms are discussed in light of the most recent progress on the entry of banda-, phlebo-, and uukuviruses, which together constitute the three prominent genera in the Phenuiviridae family.
Assuntos
Infecções por Bunyaviridae/virologia , Mamíferos/virologia , Phlebovirus/fisiologia , Internalização do Vírus , Animais , Infecções por Bunyaviridae/fisiopatologia , Endocitose , Humanos , Phlebovirus/genética , Ligação ViralRESUMO
Viruses exhibit an elegant simplicity, as they are so basic, but so frightening. Although only a few are life threatening, they have substantial implications for human health and the economy, as exemplified by the ongoing coronavirus pandemic. Viruses are rather small infectious agents found in all types of life forms, from animals and plants to prokaryotes and archaebacteria. They are obligate intracellular parasites, and as such, subvert many molecular and cellular processes of the host cell to ensure their own replication, amplification, and subsequent spread. This special issue addresses the cell biology of viral infections based on a collection of original research articles, communications, opinions, and reviews on various aspects of virus-host cell interactions. Together, these articles not only provide a glance into the latest research on the cell biology of viral infections, but also include novel technological developments.
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Viroses/patologia , Animais , Betacoronavirus/fisiologia , Interações Hospedeiro-Patógeno , Humanos , SARS-CoV-2 , Transdução de Sinais , Viroses/metabolismo , Viroses/virologia , Zika virus/fisiologiaRESUMO
Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.
Assuntos
Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Febre do Vale de Rift/metabolismo , Vírus da Febre do Vale do Rift/metabolismo , Proteínas não Estruturais Virais/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Proteínas Amiloidogênicas/química , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Núcleo Celular/virologia , Chlorocebus aethiops , Células HeLa , Humanos , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Agregação Patológica de Proteínas/metabolismo , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/patogenicidade , Células Vero , Proteínas não Estruturais Virais/química , Virulência , Fatores de VirulênciaRESUMO
The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.
Assuntos
Interferon beta/antagonistas & inibidores , Genética Reversa , Vírus da Febre do Flebótomo Napolitano/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Células A549 , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , RNA Polimerases Dirigidas por DNA/genética , Genoma Viral , Humanos , Insetos , Interferon beta/imunologia , Rim/citologia , Mutação , Vírus da Febre do Flebótomo Napolitano/imunologia , Células Vero , Proteínas Virais/genéticaRESUMO
Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) is an emerging tick-borne virus that carries a high fatality rate of 12%-50%. In-depth understanding of the SFTSV-induced pathogenesis mechanism is critical for developing effective anti-SFTS therapeutics. Here, we report transcriptomic analysis of blood samples from SFTS patients. We observe a strong correlation between inflammatory responses and disease progression and fatal outcome. Quantitative proteomic analysis of SFTSV infection confirms the induction of inflammation and further reveals virus-induced mitochondrial dysfunction. Mechanistically, SFTSV infection triggers BCL2 antagonist/killer 1 (BAK) upregulation and BAK/BCL2-associated X (BAX) activation, leading to mitochondrial DNA (mtDNA) oxidization and subsequent cytosolic release. The cytosolic mtDNA binds and triggers NLRP3 inflammasome activation. Notably, the BAK expression level correlates with SFTS disease progression and fatal outcome. These findings provide insights into the clinical features and molecular underpinnings of severe SFTS, which may aid in patient care and therapeutic design, and may also be conserved during infection by other highly pathogenic viruses.
Assuntos
Infecções por Bunyaviridae/metabolismo , DNA Mitocondrial/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Phlebovirus/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Adulto , Animais , Infecções por Bunyaviridae/genética , Infecções por Bunyaviridae/virologia , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Inflamação/genética , Interleucina-1beta/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Modelos Biológicos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 8 Toll-Like/metabolismo , Transcriptoma/genéticaRESUMO
Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus Uukuniemi/fisiologia , Animais , Antivirais/farmacologia , Infecções por Bunyaviridae/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Espectrometria de Massas , Proteômica , Piridinas/farmacologia , Quinolinas/farmacologia , Interferência de RNA , Vírus de RNA/fisiologia , Vírus Uukuniemi/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Replicação ViralRESUMO
Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that represents as a serious health threat to both domestic animals and humans. The viral protein NSs is the key virulence factor of RVFV, and has been proposed that NSs nuclear filament formation is critical for its virulence. However, the detailed mechanisms are currently unclear. Here, we generated a T7 RNA polymerase-driven RVFV reverse genetics system based on a strain imported into China (BJ01). Several NSs mutations (T1, T3 and T4) were introduced into the system for investigating the correlation between NSs filament formation and virulence in vivo. The NSs T1 mutant showed distinct NSs filament in the nuclei of infected cells, the T3 mutant diffusively localized in the cytoplasm and the T4 mutant showed fragmented nuclear filament formation. Infection of BALB/c mice with these NSs mutant viruses revealed that the in vivo virulence was severely compromised for all three NSs mutants, including the T1 mutant. This suggests that NSs filament formation is not directly correlated with RVFV virulence in vivo. Results from this study not only shed new light on the virulence mechanism of RVFV NSs but also provided tools for future in-depth investigations of RVFV pathogenesis and anti-RVFV drug screening.