Project description:Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures (Huh-7, Huh-7.5.1, Huh-7.5.1c2, R1.09, R1.10 and R2.1) R that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells (infected Huh-7, Huh-7.5.1 and Huh-7.5.1c2) and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies. Six Huh-7 derived hepatoma cell types that have different levels of susceptibility to HCV infection in cell culture are used: Huh-7, Huh-7.5.1, Huh-7.5.1c2, R1.09, R1.10 and R2.1. Of these the first three (label starting Huh are susceptible to HCV infection and the latter three (label starting R are resistant to HCV infection. All cell types are derived from Huh-7. Huh-7.5.1 is a subclone of Huh-7.5 that in turn is a subclone of Huh-7. Huh-7.5.1c2 is a subclone of Huh-7.5.1. R1.09 and R1.10 are subclones of R1 that is inturn a sublone of Huh-7.5,1. R2.1 is a subclone of Huh-7.5.1. 39 samples are used. In every case there are 3 biological replicates, i.e., there are 13 unique conditions (39/3=13). These samples are subdivided between two studies: (1) A comparison of HCV infection resistant cells R1.09, R1.10 and R2 against HCV susceptible Huh-7.5.1. (2) A comparison of JFH1 HCV infected Huh-7, Huh-7.5.1 and Huh-7.5.1c2 cells versus their uninfected counterparts. In each case there are two uninfected counterparts, cells that were harvested after being in culture for 20 hours and cells that were harvested at the same time point as the infected cells, that is, at the peak time of infection. Peak times for infection vary between cells, depending on their susceptibility to infection: 168 hours for Huh-7, 120 hours for Huh-7.5.1 and 96 hours for Huh-7.5.1c2.

Project description:Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures (Huh-7, Huh-7.5.1, Huh-7.5.1c2, R1.09, R1.10 and R2.1) R that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells (infected Huh-7, Huh-7.5.1 and Huh-7.5.1c2) and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies. Six Huh-7 derived hepatoma cell types that have different levels of susceptibility to HCV infection in cell culture are used: Huh-7, Huh-7.5.1, Huh-7.5.1c2, R1.09, R1.10 and R2.1. Of these the first three (label starting Huh are susceptible to HCV infection and the latter three (label starting R are resistant to HCV infection. All cell types are derived from Huh-7. Huh-7.5.1 is a subclone of Huh-7.5 that in turn is a subclone of Huh-7. Huh-7.5.1c2 is a subclone of Huh-7.5.1. R1.09 and R1.10 are subclones of R1 that is inturn a sublone of Huh-7.5,1. R2.1 is a subclone of Huh-7.5.1.

Project description:Purpose: Chronic infection with hepatitis B virus is the leading global risk factor for the development of liver cancer. A large body of research has shown the many effects an HBV infection has on cellular physiology, particularly on pathways that may be involved in the development of HBV-associated diseases. Unfortunately, a significant portion of this research has been done in systems that may not mimic what is seen in a primary hepatocyte, and is not done on a transcriptome-wide scale. Because of this, we performed an RNA-seq analysis of primary rat hepatocytes expressing HBV to determine the global changes HBV has on primary hepatocyte physiology. Methods: To do this RNA-seq analysis, triplicate samples of total RNA were collected from cultured primary rat hepatocytes infected with adenovirus expressing GFP alone (AdGFP) or GFP along with a greater than unit length copy of the HBV genome (AdHBV). Samples were collected either 24h or 48h after infection. cDNA libraries were sequenced two times using the Illumina HiSeq or Illumina NextSeq platform to generate either 1x50bp or 1x75bp reads. Reads from each sequencing run were mapped using the STAR aligner, and output BAMs were merged into a single BAM for each sample. The merged BAM was further analyzed in R using the GenomicAlignments package to quantify number of reads per transcript and DESeq2 to determine differential expression. Reads per kilobase transcript per million total reads (RPKM) was calculated by dividing reads per transcript by the transcript length and then normalizing to the total number of reads in the sample. Results: Following this pipeline, we were able to identify a number of HBV-mediated differentially expressed transcripts at 24h and 48h post-infection. Further pathway analysis of these differentially expressed transcripts identified many important cellular pathways, including those involved with cell cycle regulation and metabolism, as being differentially regulated by HBV in primary hepatocytes. mRNA profiles of HBV-expressing and non-expressing primary rat hepatocytes were generated, in triplicate, 24h and 48h post-infection using Illumina HiSeq 2500 and NextSeq 500 instruments.

Dataset Information

Transcriptome Analysis of Huh7 cells Infected with Adenovirus Overexpression GSTZ1 or PCK1

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