Initial HCV infection of adult hepatocytes triggers a temporally structured transcriptional program containing diverse pro- and anti-viral elements

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven donors. Coordinated sampling identified minimal gene dysregulation at six hours post infection (hpi) in PHHs. In contrast, at 72 hpi, massive increases in the breadth and magnitude of HCV-induced gene dysregulation were apparent, affecting gene classes associated with diverse biological processes. Comparison with HCV-induced transcriptional dysregulation in Huh-7.5 cells identified limited overlap between the two systems. Of note, in PHHs, HCV infection initiated broad upregulation of canonical interferon (IFN)-mediated defense programs, limiting viral RNA replication and abrogating virion release. In addition, we confirm that constitutive expression of IRF1 in PHHs maintains a steady-state antiviral program in the absence of infection which can further reduce HCV RNA replication. We also detected infection-induced signatures of translational shutoff in PHHs - downregulation of ~90 genes encoding components of the EIF2 translation initiation complex and ribosomal subunits. As HCV polyprotein translation occurs independently of the EIF2 complex, this process is pro-viral: only translation initiation of host transcripts is arrested. The combination of antiviral intrinsic and inducible immunity, balanced against pro-viral programs, including translational arrest, maintains HCV replication at a low-level in PHHs. This may ultimately keep HCV under the radar of extra-hepatocyte immune surveillance while initial infection is established, promoting tolerance, preventing clearance and facilitating progression to chronicity. Primary human hepatocytes (PHHs) were isolated from livers of seven donors undergoing partial hepatectomy. Plated PHHs and Huh-7.5 were infected with HCV (strain Jc1) or treated with conditioned medium, and incubated (6 or 72 hours) prior to RNA extraction and RNA-seq. PHH of donors 4-7 were also treated with UV-inactivated HCV. Huh-7.5 experiments were repeated three times for 6 hours and six times for 72 hours. HCV infection rates were determined using viral RT-qPCR, immunofluorescence staining of viral NS5A antigen and quantification of virion secretion via TCID50. JAK/STAT inhibitor ruxolitinib was used to rescue HCV virion replication and release in PHHs. Determination of the IRF1-regulon was achived via ecotopic expression in Huh-7.5 cells from three replicates, prior to RNA extraction and RNA-seq. HCV replication inhibitor 2’CMA was used to confirm IRF1 mediated restriction of both HCV replication and translation. All RNA-seq data generated were mapped to the Hg38 genome scaffold and raw count data were normalised (RPKM) for individual genes. For Diffferential Expression of Genes (DEG) analyses, p-values and fold changes in expression were calculated for individual gene comparisons. Genes identified in DEG analyses were used for subsequent Gene ontology analyses, Transcriptional Regulator analyses and Ingenuity Pathway Analyses.