## 2 Results
### RNA Sequencing Analysis of 5' Capped RNA Identifies Upresulated Unannotated Pol II Transcripts
RACE sequence data confirmed that the novel RNAs were indeed originating from chromosome 8 and not from the parent gene on chromosome 7. We are not aware of other reports of pseudogenes exhibiting increased expression during acute HCV infection. Effect of Gene Silencing of Selected Upregulated Genes on HCV JFH-1 Replication and Infectious Virus Production  
The significant upregulation of at least 18 host genes involved in glycoprotein production and processing, such as _FUT1_ (20-25-fold increased), was noted and raised the questions about the role of this pathway in the HCV life cycle (Supplemental Table 1). Fuosylation of viral proteins, such as  
Figure 8: Differential expression of a novel Pol II transcript during acute HCV infection. **(A)** RNA sequencing analysis of 5’ capped RNA showed a 7.5-fold increase in an unannotated RNA with a highly significant FDR of 113. The RNA transcript predicted by nscan is shown in lower part of Panel A in green [32]. **(B)** qPCR analysis showed a 2.5-fold increase in this unannotated Pol II RNA during acute HCV infection. The data are presented as in Figure 3.  
influenza hemagglutinin, or cellular glycoproteins involved in Golgi-dependent processes as viral secretion, might be essential to HCV replication and the production of mature infectious virions [34,35]. Since the mRNA of the _FUT1_ gene was so markedly upregulated, we determined if silencing of _FUT1_ would alter HCV replication in the infectious cell culture system. In addition, since _KLHDC7B_ was also highly upregulated during acute HCV infection (19-fold by RNA-seq) we also determined the effect of silencing this gene prior to HCV infection in Huh 7.5 cell cultures. _FUT1_ and _KLHDC7B_ mRNA levels were downregulated by specific siRNAs as verified by qPCR analysis. _FUT1_ mRNA was 46%, 35% and 26% of controls (cells transfected with scrambled siRNAs), and _KLHDC7B_ mRNA level was 43%, 39% and 24% at 24, 48 and 72 hours after transfection, respectively (Figure S5, Panel A). Control siRNAs did not downregulate FUT1 or KLHDC7B and had no significant cell toxicity (Figure S5, Panel B). 24 hours after siRNA transfection, cells were infected with JFH-1 (MOI = 0.5). The effect of siRNA gene silencing on HCV replication was first measured by qPCR quantification of HCV RNA as described in Experimental procedures. HCV RNA levels were reduced by 50%, 77% and 80% in _FUT1_ and by 74%, 77% and 78% in _KLHDC7B_ gene siRNA treated cells, relative to controls, at 24, 48 and 72 hours after HCV infection, respectively (Figure 9, Panel A). The effect of siRNA gene silencing of _FUT1_ and _KLHDC7B_ on the production of infectious HCV particles in cell culture was also determined. Media collected from siRNA treated and HCV RNA transfected cells, plus controls, was assayed to determine the titer of infectious virus produced in each culture. The titer of infectious HCV produced in cell cultures with _FUT1_ silenced was decreased by 27%, 50% and 50% at 24, 48 and 72 hours after HCV infection, respectively (Figure 9, Panel B). Silencing of _KLHDC7B_ decreased the titer of infectious HCV produced in cell culture by 72%, 76%, and 66% at the designated times relative to controls (Figure 9, Panel B).