## 2 Results
### RNA Sequencing (RNA-seq) Analysis of Annotated Gene Transcripts
Highly differentially expressed transcripts that were newly identified or previously unvalidated were studied by qPCR analysis of six additional replicates of JFH-1 infected Huh 7.5 cells (Figures 3-7, and Figures S1 to S4). The gene with the highest fold change (20-fold increase) in expression 48 and 72 hours after HCV infection was fucosyltransferase 1 (_FUT1_) which is involved in protein fucosylation and cell signaling (Figure 3, Panel A). A 24-fold increase in _FUT1_ mRNA levels after 72 hours of infection was confirmed by qPCR analysis (Figure 3, Panel B). Previous microarray studies also showed upregulation of this transcript in HCV JFH1-J6 infected Huh 7.5 cells [9]. Significant increases in mRNA levels of glycosyltransferase genes involved in the synthesis of protein-bound and lipid-bound oligosaccharides were also identified, indicating that regulation of glycosylation pathways are affected by acute HCV infection (Supplemental Table 1). Since HCV envelope glycoproteins are modified by N-linked glycosylation, some of these gene products may be required for viral protein processing and regulation of their function. Figure 2: Significantly differentially expressed annotated genes as determined by RNA-seq analysis of 5’ capped RNA compared to microarray analysis of poly(A)+ RNA (_p_\(>\) 0.05 and fold change of \(\geq\)2.0, [9]) after 48 (Panel A) and 72 hours of HCV infection of Huh 7.5 cells (Panel B). Figure 3: Increased expression of fucosyltransferase 1 (_FUT1_, Chr 19, – strand) during acute HCV infection. (**A**) RNA sequencing analysis of acutely HCV infected cells showed a 20.5-fold increase in _FUT1_ gene expression with a highly significant FDR of 105 (represents \(<\)0.0000000001 false positives out of 100). Gene expression is displayed as the number of 36 nt sequencing reads per kilobase of gene length per million reads (RPKMI) using the Integrated Genome Browser (IGB) (Experimental Procedures). The y-axis represents RPKMI, and x-axis represents chromosome location and gene structure. To present a better view of gene exon structure, the IGB sliced view that minimizes the size of intronic regions was used. RNA sequencing reads from a pool of three replicates of HCV infected cells at 6, 48 and 72 h are shown in red and mock-infected cells in blue. Gene structure (in kilobases), orientation and chromosomal location (gene start provided in parenthesis) are shown in the lower part of the panel next to Ref Seq. (**B**) Quantitative PCR (qPCR), although it measures only one specific region of transcript expression unlike sequencing analysis, was used to validate the RNA-seq results. Six mock infected and six HCV infected Huh 7.5 cell cultures (9.6 cm\({}^{2}\)) were analyzed by qPCR (n = 3 for each culture, total of n = 18) at each time point. qPCR analysis showed a 24.8-fold increase in mRNA at 72 hours after HCV infection. The qRT-PCR data represent the mean \(\pm\) SEM and the location of qPCR primers is indicated in the IGB schematic in Panel A. _p_-values were calculated using the Student’s t-test, and \(p\) values \(<\)0.05 were considered significant. The Kelch domain containing 7B (_KLHDC7B_) gene, encodes a protein of unknown function that contains Kelch-repeat \(\beta\)-propellers that are known to undergo a variety of binding interactions with other proteins [21]. _KLHDC7B_ was upregulated 19-fold in acutely HCV infected cells compared to controls (Figure 4, Panel A). Upregulation of this gene during acute HCV infection was not reported in previous studies [8,9,22]. The RNA-seq finding was confirmed by qPCR analysis with a fold change of 9 after 72 hours of infection (Figure 4, Panel B). **Figure 4.** Increased expression of the Kelch domain containing 7B (_KLHDC7B_, Chr 22, + strand) gene during acute HCV infection. (**A**) The RNA-seq analysis identified an 18.9-fold increase _KLHDC7B_ mRNA with an FDR value of 141.9. The gene structure, orientation and chromosomal location are shown below the sequencing reads.