Quantitative mRNA expression comparison of Hepatitis C Virus replicon (2a) on Huh7.5 cell lines
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ABSTRACT: We developed transcriptome expression assisted non-directed proteome profiling (TEAnDPP) method to investigate host-pathogen interaction. Analysis of HCV replicon induced host-cell metabolism perturbation at gene expression level. Gene enrichment analysis on DEG revealed disulfide formation related genes were significantly enriched. Based on this observation, we addminitrated thiol reactive chemical probes to visualize reactive thiol profile in live cell, and observed unique reactivity profile. Using SILAC-based quantitative profiling method, we identified 26 proteins that are labeled by iodoacetamide probes. Among these proteins, we discovered t-plastin was upregulated in APC140 cells, and its knock-down experiment showed significant HCV replication inhibition effect. In short, TEAnDPP strategy demonstrated its usefulness in host-pathogen interaction study for HCV infection.
Project description:Analysis of HCV replicon induced host-cell metabolism perturbation at gene expression level. Total RNA obtained from APC140 (stable cell line expressing HCV2a replicon) was compared to vehicle cell line (Huh7.5).
Project description:Tp80 is a novel antiviral compound. Antiviral mechanism of Tp80 is the inhibition of the viral genome replication through the recoverly of GPx2 expression downregulated by HCV infection. We used microarrays to evaluated the effect of Tp80 on the transcriptome of HCV replicon cells, compared with Non-infected host cells or non-treated HCV replicon cells.
Project description:Cyclophilin binding drugs, NIM811 and cyclosporin A (CsA), inhibit the replication of HCV replicon. We investigated the mode of action of these drugs and identified host factors essential for HCV replication in a subgenomic replicon model.
Project description:Cyclophilin binding drugs, NIM811 and cyclosporin A (CsA), inhibit the replication of HCV replicon. We investigated the mode of action of these drugs and identified host factors essential for HCV replication in a subgenomic replicon model. Experiment Overall Design: Cultured Huh7 cell were treated with CsA or NIM811 at different concentrations. Cells were harvested after 12, 24 or 48 hours. The extracted mRNA were hybridized on Affymetrix U133 Plus 2 microarrays.
Project description:Membrane-bound transcription factor CREB3L1 undergoes Regulated Intramembrane Proteolysis (RIP) in response to Hepatitis C infection. RIP activates CREB3L1 so that it can prevent the growth of HCV infected cells through the action of downstream genes. We over-expressed a truncated form of CREB3L1 that does not require RIP to enter the nucleus. Cells over-expressing this truncated form were isolated by Fluorescence Activated Cell Sorting (FACS). We used microarray to determine the downstream genes of CREB3L1 in comparison to a flow sorted empty vector control. HCV Replicon-containing cells were transfected with a CREB3L1Δ381-519 to determine the downstream genes.
Project description:Hepatitis C virus (HCV) RNA synthesis and protein expression affect cell homeostasis by modulation of gene expression. The impact of HCV replication on global cell transcription has not been fully evaluated. Thus, we analysed the expression profiles of different clones of human hepatoma-derived Huh7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system).<br><br>First, we compared the expression profile of HCV replicon clone 21-5 with both the Huh-7 parental cells and the 21-5 cured (21-5c) cells. In these latter, the HCV RNA has been eliminated by IFN-? treatment. To confirm data, we also analyzed microarray results from both the 21-5 and two other HCV replicon clones, 22-6 and 21-7, compared to the Huh-7 cells. The study was carried out by using the Applied Biosystems (AB) Human Genome Survey Microarray v1.0 which provides 31,700 probes that correspond to 27,868 human genes. A total of 15 hybridization were performed, allowing to compare transcription profiles among the following groups of cell lines:<br><br>- HCV replicon clone: 21-5 (4 hybs)<br><br>- cured HCV replicon clone: 21-5c (4 hybs)<br><br>- parental cell line: Huh-7 (4 hybs)<br><br>- other HCV replicon clones: 21-7 (2 hybs); 22-6 (1 hyb)
Project description:Virus infections induce cellular gene up and down regulation, and these changes often provide clues to cellular pathways utilized by viruses. We used microarrays to examine the transcriptional responses of cultured Drosophila S2 cells to Flock House virus (FHV) replicon induction. Experiment Overall Design: Cultured S2 cells stably transfected with either a control replicon (pS2F1fs) or an FHV RNA1 replicon (pS2F1) were induced with 1 mM copper and we measured global transcript levels at 18 h after induction using Affymetrix Drosophila Genome 2.0 microarray chips.
Project description:Dengue virus (DENV) is a major human pathogen that belongs to the flavivirus genus. Among non-structural viral proteins, NS1 is an enigmatic factor that is exclusively encoded by members of the flavivirus genus within the Flaviviridae family and accomplishes different functions during DENV infection. To gain insight into the molecular and cellular function of NS1 in viral replication, we generated a tagged NS1 DENV replicon and identified the associated host proteins during active viral replication. Replicons are self-replicating flavivirus RNAs containing large in-frame deletions in the structural genes and are useful tools to study translation and RNA amplification of several flaviviruses. To establish a global map of NS1-host protein interactions occurring during DENV replication, we stably expressed a DENV2 replicon encoding NS1 tagged with N-terminal FLAG and HA epitopes (FH-NS1) or the untagged version (WT) in three different human cell lines (Raji, HeLa and HAP1). Interactors of NS1 were identified by AP-MS/MS from these three different cell lines.
Project description:Virus infections induce cellular gene up and down regulation, and these changes often provide clues to cellular pathways utilized by viruses. We used microarrays to examine the transcriptional responses of cultured Drosophila S2 cells to Flock House virus (FHV) replicon induction.