RNA sequencing of BHK-21 cells carrying alphaviral RNA replication system
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ABSTRACT:
PROVIDER: PRJNA898802 | ENA |
REPOSITORIES: ENA
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Project description:Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. RNA-seq of 3 different cells was performed to analyze the effect of introducing different versions of RNA replication systems (i.e., repRNA-v3 and repRNA-v4) into BHK-21 cells on endogenous gene expression. Analysis of the transcriptome profiling data indicated that repRNA-v4 stimulated lower interferon signaling and may have lower cytopathicity.
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Project description:Single cell RNA-seq of BHK-21 cells carrying alphaviral RNA replication system
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Project description:Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. To determine the degradation rate of alphaviral RNA in BHK-21 cells, 10 µg of repRNA-v4 RNA was transfected into 4 million wildtype BHK-21 cells via electroporation. Samples were collected at 1-hour, 3-hour, and 12-hour time points for subsequent RNA-seq analysis. The findings revealed that the half-life of alphaviral RNA in the cells was approximately 3.5 hours.
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Project description:Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused huge economic losses to the duck industry in China since 2010. Moreover, DTMUV can also infect geese, chickens, house sparrows and replicate in in the brain, spleen, liver and kidneys of BALB/c mice, which poses public health concerns. So developing vaccines to combat DTMUV becomes urgent. Baby hamster kidney cell line (BHK-21) is usually employed to produce veterinary vaccine and DTMUV as well as other flaviviruses can be propagated in BHK-21 cells. However, information about mammalian host cell responses to DTMUV infection is limited. In this study, LC–MS/MS coupled to iTRAQ labeling was used to quantitatively identify differentially expressed cellular proteins in DTMUV-infected BHK-21 cells. We identified 192 differentially expressed cellular proteins including 11 upregulated proteins and 8 downregulated at 24 hpostinfection; 25 upregulated proteins and 151 downregulated at 48 h postinfection. Some of these proteins were involved in viral infection.
2017-05-23 | PXD005731 | Pride
Project description:Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. Toinvestigatetheexpressionheterogeneityofself-replicatingRNAsinrepRNA-v4cells,weperformedsingle-cellRNA-seqanalysisusingthe10xGenomicssequencingmethod.Ouranalysisofthesingle-cellRNA-seqprofilingdatarevealedarelativelyuniformexpressionpatternofself-replicatingRNAswithinrepRNA-v4cells.
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Project description:We used RNA-sequencing on the Illumina platform to analyze transcriptome profiles of BHK-21 acutely infected with FMDV. In total, we obtained more than 342.8 million raw sequencing reads. And approximately 336.5 million clean reads (98.14%) were acquired by filtering out the adapters and trimming ambiguous reads. We found that cellular response of BHK-21 is related to certain types of alterations in the host cell transcriptome profiles at 24 h post infection.
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Project description:Mesocricetus auratus strain:BHK-21 cells Raw sequence reads
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Project description:Measurement of the degradation rate of alphaviral RNA in BHK-21 cells
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Project description:RNA interference (RNAi) is a powerful tool to silence gene expression posttranscriptionally. In this study, we evaluated the antiviral potential of small interfering RNA (siRNA) targeting VP1 of foot-and-mouth disease virus (FMDV), which is essential during the life cycle of the virus and plays a key role in virus attachment to susceptible cells. We investigated in vivo the inhibitory effect of VP1-specific siRNAs on FMDV replication in BHK-21 cells and suckling mice, a commonly used small animal model. The results showed that transfection of siRNA-expressing plasmids gave an 80 to 90% reduction in the expression of FMDV VP1 in BHK-21 cells. Moreover, BHK-21 cells transiently transfected with siRNA-expressing plasmids were specifically resistant to FMDV infection when exposed to 100 50% tissue culture infective doses of virus, and the antiviral effects extended to almost 48 h postinfection. Furthermore, subcutaneous injection of siRNA-expressing plasmids in the neck made suckling mice significantly less susceptible to FMDV. In conclusion, our data suggests that RNAi may provide a viable therapeutic approach to treat FMDV infection.
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Project description:BackgroundEncephalomyocarditis virus, member of Cardiovirus genus within Picornaviridae family, is an important pathogen that infects different domestic and wild animals. However, the molecular mechanism of its entry remains unclear. In this study, we investigated the mechanism of EMCV infectivity in relation to endocytic pathway using BHK-21 cells.MethodsThe function of numerous cellular key factors implicated in the various endocytic mechanisms were systematically explored using chemical inhibitors. Furthermore, RNA interference (RNAi) as well as the overexpression of dominant protein combined to virus infectivity assays, and confocal microscopy was used to examine EMCV infection in details.ResultsThe results indicated that the EMCV entry into BHK-21 cells depends on caveolin, dynamin, and actin but not clathrin nor macropinocytosis pathways. The effects of overexpression and knockdown of caveolin-1, one components of the caveolae, was examined on EMCV infection. The results showed that EMCV infection was positive correlation with caveolin-1 expression. Confocal microscopy analysis and internalization assay showed that caveolin-1 is required at the early stage of EMCV infection.ConclusionsCaveolin-1, dynamin, and actin-dependent endocytosis pathways are necessary for EMCV infection in vitro.
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