## 3 Discussion  
We describe the first report of RNA sequencing analysis of 5' capped RNAs isolated from acutely HCV infected Huh 7.5 cells and provide evidence that this approach identifies many new annotated and unannotated genes that are differentially expressed during acute HCV infection. At 48 and 72 hours post infection more than 80% of significantly differentially expressed genes reported in our study were not identified in prior gene array analysis of acute HCV infection using the same fold change. We also present the first analysis of unannotated transcripts, some likely to be ncRNAs, that are differentially expressed during acute HCV infection and may play significant roles in the viral-host interactions. Follow up siRNA studies of two newly identified, highly upregulated genes, _FUT1_ and _KLHDC7B_, provided evidence their expression during acute HCV infection is essential for the production of HCV RNA and infectious viral particles. Our results suggest that this general approach will have applications in studying viral-host interactions in both model systems and clinical biospecimens. Our studies were done in proliferative, standard cultures, of Huh 7.5 cells. It will be of interest to determine the similarities and differences in gene expression of acutely HCV infected proliferative and growth arrested differentiated human hepatoma-derived cells [36]. In comparison with our reported ENCODE tiling array (interrogates 1% of the human genome) analysis of HCV infected cirrhotic liver that identified 95 differentially expressed genes as compared to controls, our current analysis of acutely infected hepatoma cells showed that 20% of these genes were also differentially expressed [14]. This is a relatively large percentage of overlap since liver represents a much more complex transcriptome due to the presences of a number of other cell types besides hepatocytes and the fact that the ENCODE analysis was of chronically infected cirrhotic liver. Similar changes in genes involved in the immune response (e.g., _IRF1_, _RFX5_ and _PSMB4_), cell structure (_FLNA_, _PLXNA3_ and _CAPZA_) and growth (_CTGF_ and _RHBDF1_) were observed in the prior and current studies, providing evidence that there is overlap in the host response to acute and chronic HCV infection and that the Huh 7.5 model can partly mimic _in vivo_ processes occurring during HCV infection. The results also indicate that transcriptome changes in acutely infected Huh 7.5 cells become more profound with propagation of infection. This suggests that as a greater percentage of cells becomes infected that the biological effects become more pronounced, raising interesting questions regarding the pathobiology in selected patients with hepatitis C.  
**Figure 9**. siRNA silencing of _FUT1_ and _KLHDC7B_ inhibit HCV replication and infectious virus production. Huh 7.5 cells were transfected with control siRNA (white bars), _FUT1_ (gray bars) or _KLHDC7B_ (black bars) and infected with JFH-1 (MOI = 0.5) for two hours, supplemented with fresh complete DMEM and continuously cultured to 24, 48, and 72 hours. At each time point, HCV RNA was measured by real-time PCR (**A**) and the titer of infectious HCV was measured by serial dilution of culture supernatants (**B**). The percent inhibition produced by each siRNA is shown above each bar in the graph.