Ultrastructural Characterization of Zika Virus Replication Factories.
Ontology highlight
ABSTRACT:
SUBMITTER: Cortese M
PROVIDER: S-EPMC5340982 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Plus-stranded RNA viruses replicate in the cytosol of infected cells, in membrane-bound replication complexes containing the replicase proteins, the viral RNA and host proteins. The formation of the replication and transcription complexes (RTCs) through the rearrangement of cellular membranes is currently being actively studied for viruses belonging to different viral families. In this work, we identified double-membrane vesicles (DMVs) in the cytoplasm of cells infected with the equine torovirus Berne virus (BEV), the prototype member of the Torovirus genus (Coronaviridae family, Nidovirales order). Using confocal microscopy and transmission electron microscopy, we observed a close relationship between the RTCs and the DMVs of BEV. The examination of BEV-infected cells revealed that the replicase proteins colocalize with each other and with newly synthesized RNA and are associated to the membrane rearrangement induced by BEV. However, the double-stranded RNA, an intermediate of viral replication, is exclusively limited to the interior of DMVs. Our results with BEV resemble those obtained with other related viruses in the Nidovirales order, thus providing new evidence to support the idea that nidoviruses share a common replicative structure based on the DMV arranged clusters.
| S-EPMC7162420 | biostudies-literature
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus that replicates in both vertebrate and insect cells, whereas insect-specific flaviviruses (ISF) replicate only in insect cells. We sought to convert ZIKV, from a dual-tropic flavivirus, into an insect-specific virus for the eventual development of a safe ZIKV vaccine. Reverse genetics was used to introduce specific mutations into the furin cleavage motif within the ZIKV pre-membrane protein (prM). Mutant clones were selected, which replicated well in C6/36 insect cells but exhibited reduced replication in non-human primate (Vero) cells. Further characterization of the furin cleavage site mutants indicated they replicated poorly in both human (HeLa, U251), and baby hamster kidney (BHK-21) cells. One clone with the induced mutation in the prM protein and at positions 291and 452 within the NS3 protein was totally and stably replication-defective in vertebrate cells (VSRD-ZIKV). Preliminary studies in ZIKV sensitive, immunodeficient mice demonstrated that VSRD-ZIKV-infected mice survived and were virus-negative. Our study indicates that a reverse genetic approach targeting the furin cleavage site in prM can be used to select an insect-specific ZIKV with the potential utility as a vaccine strain.
| S-EPMC7733535 | biostudies-literature
Project description:RNA polymerase II (RNAPII) transcription has been proposed to occur at transcription factories; nuclear focal accumulations of the active, phosphorylated forms of RNAPII. The low ratio of transcription factories to active genes and transcription units suggests that genes must share factories. Our previous analyses using light microscopy have indicated that multiple genes could share the same factory. Furthermore, we found that a small number of specialized transcription factories containing high levels of the erythroid-specific transcription factor KLF1 preferentially transcribed a network of KLF1-regulated genes. Here we used correlative light microscopy in combination with energy filtering transmission electron microscopy (EFTEM) and electron microscopy in situ hybridization (EMISH) to analyse transcription factories, transcribing genes, and their nuclear environments at the ultrastructural level in ex vivo mouse foetal liver erythroblasts. We show that transcription factories in this tissue can be recognized as large nitrogen-rich structures with a mean diameter of 130 nm, which is considerably larger than that previously seen in transformed cultured cell lines. We show that KLF1-specialized factories are significantly larger, with the majority of measured factories occupying the upper 25th percentile of this distribution with an average diameter of 174 nm. In addition, we show that very highly transcribed genes associated with erythroid differentiation tend to occupy and share the largest factories with an average diameter of 198 nm. Our results suggest that individual factories are dynamically organized and able to respond to the increased transcriptional load imposed by multiple highly transcribed genes by significantly increasing in size.
| S-EPMC3215576 | biostudies-literature
Project description:During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells.Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.
| S-EPMC3993787 | biostudies-other
Project description:Proteomic analysis of isolated viral factories along a timeline after infection of the amoeba Acanthamoeba polyphaga by the giant Mimivirus
2018-10-26 | PXD004203 | Pride
Project description:The Zika virus (ZIKV) outbreak in 2016 in South America with specific pathogenic outcomes highlighted the need for new antiviral substances with broad-spectrum activities to react quickly to unexpected outbreaks of emerging viral pathogens. Very recently, the natural compound silvestrol isolated from the plant Aglaia foveolata was found to have very potent antiviral effects against the (-)-strand RNA-virus Ebola virus as well as against Corona- and Picornaviruses with a (+)-strand RNA-genome. This antiviral activity is based on the impaired translation of viral RNA by the inhibition of the DEAD-box RNA helicase eukaryotic initiation factor-4A (eIF4A) which is required to unwind structured 5´-untranslated regions (5'-UTRs) of several proto-oncogenes and thereby facilitate their translation. Zika virus is a flavivirus with a positive-stranded RNA-genome harboring a 5'-capped UTR with distinct secondary structure elements. Therefore, we investigated the effects of silvestrol on ZIKV replication in A549 cells and primary human hepatocytes. Two different ZIKV strains were used. In both infected A549 cells and primary human hepatocytes, silvestrol has the potential to exert a significant inhibition of ZIKV replication for both analyzed strains, even though the ancestor strain from Uganda is less sensitive to silvestrol. Our data might contribute to identify host factors involved in the control of ZIKV infection and help to develop antiviral concepts that can be used to treat a variety of viral infections without the risk of resistances because a host protein is targeted.
| S-EPMC5923443 | biostudies-literature
Project description:The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.
| S-EPMC6854498 | biostudies-literature
Project description:Zika virus (ZIKV) has emerged globally as an important pathogen, since it has been recognized as a cause of microcephaly and other neurologic processes and sequalae in newborns. The virus shares homology with Hepaciviruses and therefore may be a cause of hepatitis. We sought to characterize ZIKV replication in hepatocyte-derived cell lines. Huh7.5 and HepG2 cells were infected with ZIKV and replication potential was evaluated by multiple methods including plaque assay, qRT-PCR, negative-strand ZIKV RNA production, and ZIKV NS1 protein production. Growth curves in cells and supernatant were compared to replicative capacity in Vero cells. Overall, viral replication in both hepatocyte lines approximated that observed in the Vero cells. Cell cytopathology was observed after 3 days of infection and apoptosis markers increased. Transmission electron microscopy revealed evidence of viral capsids in cells and negative staining revealed ZIKV particles in the supernatant. Conclusions: Hepatocyte-derived cell lines are permissive for ZIKV replication and produce an overt cytopathic effect consistent with development of an acute viral hepatitis. Further evaluation of replication and injury is warranted.
| S-EPMC6426209 | biostudies-literature
Project description:The Zika virus (ZIKV) is primarily transmitted via an infected mosquito bite, during sexual intercourse, or in utero mother to child transmission. When a fetus is infected, both neurological malformations and deficits in brain development are frequently manifested. As such, there is a need for vaccines or drugs that may be used to cure ZIKV infections. Metabolic pathways play a crucial role in cell differentiation and development. More importantly, polyamines play a key role in replication and translation of several RNA viruses, including ZIKV, Dengue virus, and Chikungunya virus. Here, we present polyamine analogues (BENSpm and PG11047) and their corresponding polymer prodrug derivatives for inhibiting ZIKV infection by intersecting with polyamine catabolism pathways. We tested the compounds against ZIKV African (MR766) and Asian (PRVABC59) strains in human kidney epithelial (Vero) and glioblastoma derived (SNB-19) cell lines. Our results demonstrate potent inhibition of ZIKV viral replication in both cell lines tested. This antiviral effect was mediated by the upregulation of two polyamine catabolic enzymes, spermine oxidase, and spermidine (SMOX)/spermine N1-acetyltransferase (SAT1) as apparent reduction of the ZIKV infection following heterologous expression of SMOX and SAT1. On the basis of these observations, we infer potential use of these polyamine analogues to treat ZIKV infections.
| S-EPMC6538062 | biostudies-literature
Project description:Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.
| S-EPMC5699088 | biostudies-other