RESUMEN
Tupanviruses, members of the family Mimiviridae, infect phagocytic cells. Particle uncoating begins inside the phagosome, with capsid opening via the stargate. The mechanism through which this opening takes place is unknown. Once phagocytized, metal ion flux control and ROS are induced to inactivate foreign particles, including viruses. Here, we studied the effect of iron ions, copper ions, and H2O2 on Tupanvirus particles. Such treatments induced stargate opening in vitro, as observed by different microscopy techniques. Metal-treated viruses were found to be non-infectious, leading to the hypothesis that stargate opening likely resulted in the release of the viral seed, which is required for infection initiation. To the best of our knowledge, this is the first description of a giant virus capsid morphological change induced by transition metals and H2O2, which may be important to describe new virulence factors and capsid uncoating mechanisms.
Asunto(s)
Peróxido de Hidrógeno , Oxidación-Reducción , Replicación Viral , Peróxido de Hidrógeno/farmacología , Mimiviridae/fisiología , Mimiviridae/genética , Cápside/metabolismo , Cobre/farmacología , Cobre/metabolismo , Hierro/metabolismo , Animales , Desencapsidación ViralRESUMEN
The virosphere is fascinatingly vast and diverse, but as mandatory intracellular parasites, viral particles must reach the intracellular space to guarantee their species' permanence on the planet. While most known viruses that infect animals explore the endocytic pathway to enter the host cell, a diverse group of ancient viruses that make up the phylum Nucleocytoviricota appear to have evolved to explore new access' routes to the cell's cytoplasm. Giant viruses of amoeba take advantage of the phagocytosis process that these organisms exploit a lot, while phycodnavirus must actively break through a algal cellulose cell wall. The mechanisms of entry into the cell and the viruses themselves are diverse, varying in the steps of adhesion, entry, and uncoating. These are clues left by evolution about how these organisms shaped and were shaped by convoluting with eukaryotes.
Asunto(s)
Virus Gigantes/fisiología , Internalización del Virus , Amoeba/virología , Animales , Coevolución Biológica , Chlorella/virología , Virus Gigantes/clasificación , Acoplamiento Viral , Desencapsidación ViralRESUMEN
Although human astroviruses (HAstVs) are important agents of gastroenteritis in young children, the studies aimed at characterizing their biology have been limited, in particular regarding their cell entry process. It has been shown that HAstV serotype 8 enters human cells by a classical clathrin-mediated endocytosis pathway; however, the cell receptor or other cell entry factors that may be relevant for an efficient viral infection are unknown. In this work we used a far-Western blotting approach to identify cellular proteins that interact with the recombinant capsid spike proteins of HAstV serotypes 1, 2, and 8, synthesized in Escherichia coli. We identified the 72 kDa protein disulfide isomerase A4 (PDIA4) as a binding partner for HAstV-1 and -8 spikes, but not for the HAstV-2 spike. In agreement with this observation, the PDI inhibitor 16F16 strongly blocked infection by HAstV serotypes 1 and 8, but not serotype 2. RNA interference of PDIA4 expression selectively blocked HAstV-8 infectivity. We also showed that the PDI activity does not affect virus binding or internalization but is required for uncoating of the viral genome.
Asunto(s)
Infecciones por Astroviridae/metabolismo , Infecciones por Astroviridae/virología , Interacciones Huésped-Patógeno , Mamastrovirus/fisiología , Proteína Disulfuro Isomerasas/metabolismo , Desencapsidación Viral , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Células Cultivadas , Humanos , Mamastrovirus/efectos de los fármacos , Unión Proteica , Internalización del VirusRESUMEN
Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus.IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimivirus and provide new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating, and morphogenesis. Furthermore, we demonstrate that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, allow us to present an ultimate model for the mimivirus replication cycle.
Asunto(s)
Acanthamoeba castellanii/virología , Mimiviridae/fisiología , Internalización del Virus , Replicación Viral/fisiología , Desencapsidación Viral/fisiología , Acanthamoeba castellanii/metabolismo , FagocitosisRESUMEN
UNLABELLED: The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. IMPORTANCE: Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade regarding molecular aspects of the fusion step, but little is known about the events that follow this process, which leads to viral RNA release from the nucleocapsid. Here, we investigated the fate of nucleocapsid components (capsid protein and viral genome) during the infection process and found that capsid is degraded by the ubiquitin-proteasome system. However, in contrast to that observed for other RNA and DNA viruses, dengue virus capsid degradation was not responsible for genome uncoating. Interestingly, we found that dengue virus genome release requires a nondegradative ubiquitination step. These results provide the first insights into dengue virus uncoating and present new opportunities for antiviral intervention.
Asunto(s)
Cápside/metabolismo , Virus del Dengue/genética , Genoma Viral , Desencapsidación Viral , Células A549 , Animales , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Cricetinae , Dengue , Virus del Dengue/fisiología , Humanos , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Viral/genética , Ubiquitinación , Virión , Internalización del Virus , Liberación del VirusRESUMEN
Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.