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BACKGROUND: Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES: In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS: Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS: The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS: Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.
Assuntos
Acanthamoeba castellanii , Efeito Citopatogênico Viral , Mimiviridae , Mimiviridae/fisiologia , Cinética , Acanthamoeba castellanii/virologiaRESUMO
BACKGROUND Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.
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The Salmonella enterica bacteriophage P22 is one of the most promising models for the development of virus-like particle (VLP) nanocages. It possesses an icosahedral T = 7 capsid, assembled by the combination of two structural proteins: the coat protein (gp5) and the scaffold protein (gp8). The P22 capsid has the remarkable capability of undergoing structural transition into three morphologies with differing diameters and wall-pore sizes. These varied morphologies can be explored for the design of nanoplatforms, such as for the development of cargo internalization strategies. The capsid proteic nature allows for the extensive modification of its structure, enabling the addition of non-native structures to alter the VLP properties or confer them to diverse ends. Various molecules were added to the P22 VLP through genetic, chemical, and other means to both the capsid and the scaffold protein, permitting the encapsulation or the presentation of cargo. This allows the particle to be exploited for numerous purposes-for example, as a nanocarrier, nanoreactor, and vaccine model, among other applications. Therefore, the present review intends to give an overview of the literature on this amazing particle.
Assuntos
Bacteriófago P22 , Viroides , Capsídeo , Proteínas do Capsídeo/genética , Núcleo Celular , NanotecnologiaRESUMO
Almost two decades after the isolation of the first amoebal giant viruses, indubitably the discovery of these entities has deeply affected the current scientific knowledge on the virosphere. Much has been uncovered since then: viruses can now acknowledge complex genomes and huge particle sizes, integrating remarkable evolutionary relationships that date as early as the emergence of life on the planet. This year, a decade has passed since the first studies on giant viruses in the Brazilian territory, and since then biomes of rare beauty and biodiversity (Amazon, Atlantic forest, Pantanal wetlands, Cerrado savannas) have been explored in the search for giant viruses. From those unique biomes, novel viral entities were found, revealing never before seen genomes and virion structures. To celebrate this, here we bring together the context, inspirations, and the major contributions of independent Brazilian research groups to summarize the accumulated knowledge about the diversity and the exceptionality of some of the giant viruses found in Brazil.
Assuntos
Amoeba/virologia , Vírus Gigantes/genética , Vírus Gigantes/isolamento & purificação , Virologia/história , Biodiversidade , Brasil , Ecossistema , Genoma Viral , Vírus Gigantes/classificação , Vírus Gigantes/ultraestrutura , História do Século XXI , FilogeniaRESUMO
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.
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Acanthamoeba castellanii/metabolismo , Fungos/patogenicidade , Lectina de Ligação a Manose/metabolismo , Acanthamoeba castellanii/química , Acanthamoeba castellanii/microbiologia , Acanthamoeba castellanii/ultraestrutura , Animais , Candida albicans/patogenicidade , Candida albicans/ultraestrutura , Concanavalina A/metabolismo , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Histoplasma/patogenicidade , Histoplasma/ultraestrutura , Interações Hospedeiro-Patógeno , Larva/microbiologia , Lepidópteros/microbiologia , Manose/química , Manose/metabolismo , Lectina de Ligação a Manose/química , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Saccharomyces cerevisiae/patogenicidade , Saccharomyces cerevisiae/ultraestrutura , Fatores de Tempo , Imagem com Lapso de Tempo , Virulência , Fatores de Virulência/metabolismoRESUMO
Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.
Assuntos
Acanthamoeba castellanii/metabolismo , Amebíase/parasitologia , Vesículas Extracelulares/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba castellanii/genética , Animais , Linhagem Celular , Vesículas Extracelulares/genética , Homeostase , Humanos , Transporte Proteico , Proteoma/genética , Proteômica , Proteínas de Protozoários/genética , Via SecretóriaRESUMO
The inclusion of Mimiviridae members in the putative monophyletic nucleocytoplasmic large DNA virus (NCLDV) group is based on genomic and phylogenomic patterns. This shows that, along with other viral families, they share a set of genes known as core or "hallmark genes," including the gene for the major capsid protein (MCP). Although previous studies have suggested that the maturation of mimivirus MCP transcripts is dependent on splicing, there is little information about the processing of this transcript in other mimivirus isolates. Here we report the characterization of a new mimivirus isolate, called Kroon virus (KV) mimivirus. Analysis of the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates revealed a remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. In addition, sequencing of KV and Acanthamoeba polyphaga mimivirus (APMV) MCP transcripts has shown that inside the family, even related giant viruses may present different ways to process the MCP mRNA. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.IMPORTANCE Mimivirus isolates have been obtained by prospecting studies since 2003. Based on genomic and phylogenomic studies of conserved genes, these viruses have been clustered together with members of six other viral families. Although the major capsid protein (MCP) gene is an important member of the so-called "hallmark genes," there is little information about the processing and structure of this gene in many mimivirus isolates. In this work, we have analyzed the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates; these genes showed remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.
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Proteínas do Capsídeo/genética , Evolução Molecular , Regulação Viral da Expressão Gênica , Mimiviridae/genética , Splicing de RNA , Transcrição Gênica , Genoma Viral , Mimiviridae/classificação , Mimiviridae/isolamento & purificação , Mimiviridae/ultraestrutura , Filogenia , RNA Viral , Replicação Viral , Microbiologia da ÁguaRESUMO
UNLABELLED: Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the â¼ 1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE: APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.
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Acanthamoeba/virologia , Glicoproteínas/metabolismo , Mimiviridae/fisiologia , Proteínas Virais/metabolismo , Ligação Viral , Acanthamoeba/fisiologia , Acanthamoeba/ultraestrutura , Acetilglucosamina/metabolismo , Análise de Variância , Manose/metabolismo , Microscopia Eletrônica de Transmissão , Especificidade da Espécie , Replicação Viral/fisiologiaRESUMO
The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.