Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes
Long term cell culture adaptation of hepatitis C virus resulted in increased replication fitness in various human liver cell lines but in a moderate decrease in virus particle production upon infection of primary human hepatocytes (PHH). In order to identify molecular mechanisms conferring phenotypic differences in replicative fitness of the cell culture adapted virus strain p100pop, we infected PHH and Huh-7 cells with HCV, using the cell culture adapted strain p100pop or a Jc1 strain with similar genome organisation (Jc1-SP). Total RNA was extracted at 28 hours post inoculation and used for RNA sequencing. Transcriptome analyses, gene ontology enrichment analyses and KEGG pathway analyses revealed strong differences in the transcriptional signature of infected hepatoma cells and primary hepatocytes and the two virus strains used in this study. Wheras an innate immune response was induced in primary cells regardless of the infecting virus strain, this was not detectable in Huh-7 cells. Even though both viruses induce a similar host response in primary cells, the data indicate that the presence of cell culture adaptive mutations results in an increased expression of genes involved in the defense response to viral infection. In Huh-7 cells, differentially expressed genes associated with ER stress and apoptosis were solely enhanced upon p100pop infection but not upon Jc1-SP infection. Overall design: Three biological independent passages of Huh-7 cells and primary human hepatocytes from  three independent donors were inoculated with HCV (Jc1-SP or p100pop). Apart from one PHH donor, hepatocytes from the same passage or donor were treated with conditioned medium and served as an uninfected control. The other  conditioned medium treated control derives from a different donor as the ones used for the infection experiments. At 28 hours post inoculation total RNA was extracted from cell lysates. Infection rates were determined using a HCV-probe based real time-quantitative PCR before RNA-sequencing was performed.  The resulting raw FASTQ files were aligned against the human reference genome GRh38 (version from 24.11.2021). Differentially expressed genes were identified by filtering on average expression for false discovery rate (FDR) correction by testing for differntilly expression due to infection while controlling for passage number of Huh-7 cells or the primary human hepatocyte donor. For the Gene Ontology enrichment analyses only differentially expressed genes with a mean RPKM value >0.5, fold change > 1.5 and FDR p-value<0.05 were considered. Further processing of these data for Gene set enrichment as well as KEGG pathway analyses were performed in R using the gseKEGG function from the RNA package 'clusterProfiler' v4.7.1.001.