## 2 Results
### RNA Sequencing (RNA-seq) Analysis of Annotated Gene Transcripts
This example shows one benefit of analyzing 5' capped by RNA sequencing and provides evidence that an isoform of _KLHDC7B_ mRNA with a longer 5' untranslated region (UTR) is expressed in Huh 7.5 cells after infection with HCV. There was no annotated gene or EST on the minus strand in this genomic region that provided an alternative explanation for the sequence reads observed upstream of the _KLHDC7B 5'_ end. (**B**) qPCR analysis verified a major increase in expression of _KLHDC7B_ and showed a 9-fold increase mRNA with the PCR primers used. Indicating a major reprogramming of host cell gene expression, a total of 276 genes involved in gene transcription were differentially expressed by 72 hours of acute HCV infection. Eighty percent of these genes were not previously determined differentially regulated during acute HCV infection. This included the upregulation of transcription factors regulating cell differentiation, inflammation and signal transduction including krupple-like factor 4 (_KLF4_, 15-fold), hexokinase domain containing 1 (_HKDC1_, 15-fold), ankyrin repeat domain 1 (_ANKRD1_, 12-fold), v-myc myelocytomatosis viral oncogene (_MYC_, 5-fold), and AT-hook transcription factor (_AKNA_, 4-fold) (Table 1). The upregulation of _AKNA_, _MYC_, and _ANKRD1_ mRNA transcripts was confirmed by qPCR (Figure 5 and Supplemental Figures 1 and 2, respectively). Two of these genes, not reported to be upregulated in previous studies, were _KLF4_ and _AKNA_. The AT-hook transcription factors, of which _AKNA_ is a member, repress or activate gene networks controlling inflammation, development, carcinogenesis, and metabolism are thought be critical in some disease states [23,24]. The finding of upregulation of _AKNA_ expression raises the question that either the host or virus uses this change to downregulate a network of inflammatory genes during acute HCV infection. Figure 5: Increased expression of AT-hook transcription factor (_AKNA_, Chr 9, – strand) during acute HCV infection. (**A**) RNA sequencing analysis showed a 4.5-fold increase with an FDR of 773.3 and (**B**) qPCR showed a 4.3-fold increase in _AKNA_ mRNA at 72 hr after HCV infection. Our RNA sequencing analysis identified another biologically unpredicted pair of genes that were activated during acute HCV infection. Inhibin beta isoforms A and E (_INHBA_ and _INHBE_) were upregulated 14 and 6-fold after acute HCV infection (Table 1, Figure 6). Upregulation of _INHBE_ was confirmed increased by qPCR analysis with an average fold change of 7 (Figure 6, Panel B). Inhibin beta E (_INHBE_) is a member of the transforming growth factor (TGF-\(\beta\)) super family and plays a role in cell growth and proliferation [25]. _INHBE_ is involved in regulation of human reproductive hormones and is dysregulated in endometrial and cervical carcinoma [25, 26]. The upregulation of _INHBE_ suggests that nonclassical TGF-\(\beta\) family member genes play regulatory roles during acute HCV infection. A number of studies have demonstrated the formation of membranous webs that include HCV replication complex proteins, during HCV infection [27, 28]. Our RNA sequencing analysis revealed a 6-fold increase in _filamin C (FLNC)_ mRNA, which produces a gene product that is critical in cross-linking actin filaments and attaching membrane glycoproteins proteins to the actin cytoskeleton (Supplemental Figure 3). Although increased _FLNC_ was previously reported, we identified 128 additional members of the cytoskeleton gene family that were significantly differentially expressed during acute HCV infection. Ninety-six of these genes were not previously identified, including upregulation of _APS_, _SLC1A4_, _PSEN2_, _MAP1B_, and _PXK_ (Supplemental Table 1). This data supports  
Figure 6: Increased expression of inhibin beta E (_INHBE_, Chr 12, + strand) during acute HCV infection.