We performed transcriptome profiling using U133A Affymetrix high-density oligonucleotide arrays on RNA samples prepared from Huh-7 cells bearing HCV replicons in the presence of 25-HC, an inhibitor of the mevalonate pathway. The transcriptional profiling was carried out using cRNA prepared from four treatment groups: (1) naïve Huh-7 cells; (2) naïve Huh-7 cells treated with 25-HC; (3) Huh-7 cells transiently transfected with pFK-I389
neo/NS3-3'/5.1 or replicon RNA from pFK-I389
neo/NS3-3'/Δ5B replicon RNAs (Fig. ); and () Huh-7 cells transiently transfected with pFK-I389
neo/NS3-3'/5.1 or pFK-I389
neo/NS3-3'/Δ5B replicon RNAs and treated with 25-HC as indicated in Fig. . The experiments were performed analogously to previous studies of antiviral small molecules on HCV replication [25
]. As previously reported [8
], the treatment of Huh-7 cells bearing HCV replicons with 25-HC caused a dose-dependent decrease in HCV replication at non-toxic concentrations as measured by firefly luciferase activity in Huh-7 cells transfected with HCV replicon RNA from pFK-I389
luc/NS3-3'/5.1 (Fig. ).
Figure 2 HCV replicons and their use in transcriptional profiling with 25-hydroxycholesterol treatment. (a) HCV genome and HCV subgenomic replicon constructs used in this study. (b) Experimental design for Affymetrix GeneChip experiments using HCV luc/NS3-3'/5.1 (more ...)
Figure 4 Gene expression patterns for PROX1, INSIG-1, NK4, and UBD in HCV- infected chimpanzees. Expression patterns of genes that are differentially regulated in HCV replicon cells during a time course of HCV infection for three infected chimpanzees displaying (more ...)
Comparisons were conducted between each set of duplicate data to determine which genes were differentially expressed in 25-HC-treated Huh-7 cells and in the 25-HC-treated HCV replicon cells. The ratios of average expression for 25-HC treated Huh-7 cells/naïve Huh-7 cells were used to compute the fold change values for the 25-HC treated column Table . Similarly the 25-HC-treated HCV replicon column in Table was computed from the ratio of average expression values from (replicon + 25-HC)/replicon samples. Transcriptome profiling of Huh-7 cells treated with 25-HC demonstrated that 69 genes were differentially regulated. Forty-seven genes were downregulated, 16 of which are clearly related to the mevalonate pathway (Table ). A number of membrane proteins and oligonucleotide-binding proteins also appear to be downregulated (Table ). By contrast, only 22 upregulated genes were observed upon treatment with 25-HC (Table ). None of these genes are directly involved in the mevalonate pathway and there appears to be no functional relationship between the entries within Table . These observations suggest that 25-HC acts at the transcriptional level as a negative regulator of fatty acid and cholesterol biosynthesis [8
], and that other changes in gene expression observed occur as secondary effects of 25-HC treatment. It is interesting to note that Huh-7 cells treated with both HCV replicon RNA and 25-HC did not show identical transcriptional profiles to cells treated analogously without HCV replicon RNA or to those only treated with HCV replicon RNA or replication-defective replicon RNA (pFK-I389
neo/NS3-3'/Δ5B replicon RNA). Specifically, 5 genes were uniquely upregulated and 22 genes were uniquely downregulated in the 25-HC-treated HCV replicon harbouring cells (Table ). Virtually all the genes in Table are hypothetical transcripts or genes of unknown function. Collectively the global gene expression changes represented by Tables , , , represent the transcriptional program that leads to an antiviral state within the host cell hepatocyte, at least for the subgenomic HCV replicon. One or more of these genes may serve as prognostic markers for HCV treatment. Therefore, we performed further studies to investigate the importance of the observed gene expression changes in Tables , , in other models for HCV pathogenesis.
GeneChip analysis of downregulated genes after 25-HC treatment of Huh-7 cells and Huh-7 cells bearing HCV subgenomic replicons.a
GeneChip analysis of upregulated genes after 25-HC treatment of Huh-7 cells and Huh-7 cells bearing HCV subgenomic replicons.a
GeneChip analysis of gene expression changes after 25-HC treatment in HCV replicons.a
We examined the transcriptional profiles of the genes from this study in three previously studied chimpanzees that were infected with HCV resulting in different outcomes of infection [28
] and for whom genome-wide transcriptional analyses were performed during the course of infection [11
]. Briefly, one chimpanzee successfully cleared the virus (sustained clearance, SC), one became persistently infected but initially transiently cleared the virus (transient clearance, TC), and the third chimpanzee developed an unrestrained persistent HCV infection (persistence, PS) in the absence of both an intrahepatic antiviral immune response and IFN-γ induction [28
]. Genome-wide transcriptional profiling did suggest that the induction of both immunological and hepatocellular genes can influence the course and outcome of HCV infection [11
]. Both the SC and TC chimpanzees displayed an altered lipid metabolism profile during the initial rise in viremia following inoculation with HCV as revealed by gene expression profiling, whereas the PS chimpanzee did not [11
To determine which genes from our study were most relevant in the context of HCV infection in the chimpanzee model, we compared genes identified in Tables , , with those of the chimpanzee data sets described previously [11
]. To this end, we compared the replicon data sets (transient transfection) with the most representative chimpanzee data sets, those containing genes that changed significantly during the initial rise in viremia (described in the methods section), since both involve an initial host response to viral RNA and both involve the modulation of lipid metabolism genes. From the 69 differentially regulated genes identified here and the 257 genes derived from transcriptome profiling of infected chimpanzees during the initial rise in viremia [11
], we identified 54 overlapping genes. These genes were further analyzed based on outcome-specific gene expression profiles, fold-induction in chimpanzees during the initial rise in viremia, function as determined by bioinformatically and using the SymAtlas at http://symatlas.gnf.org/SymAtlas
, and using the Ingenuity interaction software. The four genes selected for further study were prospero-related homeobox 1 (PROX1), insulin induced gene 1 (INSIG1), natural killer cell transcript 4 (NK4), and diubiquitin (UBD), Table .
Comparative gene expression analysis of 25-HC-treated and HCV replicon-harboring Huh-7 cells with the changes in gene expression during HCV infection in chimpanzees with different outcomes of infection.a
The PROX1 gene is a transcription factor expressed in the adult human liver that is involved in hepatocyte specification, proliferation, differentiation, and migration [29
]. It is also involved in control of bile acid synthesis and cholesterol homeostasis in vivo
]. Since HCV replication has been demonstrated to be linked with cellular lipid metabolism, and the PROX1 gene was found to have interesting expression patterns in previous gene expression studies carried out in chimpanzee [11
], the PROX1 gene was chosen for further validation in the HCV replicon cells. In the replicon data set, PROX1 was downregulated in HCV replicon cells compared with naïve Huh-7 cells (Table ). The PROX1 gene is downregulated in replicon-harbouring cells compared to naïve Huh-7 cells and represents a class of genes that may be negatively regulated during HCV replication. Interestingly, PROX1 is upregulated in TC and SC during the initial rise in viremia, but negatively regulated in PS (Table ).
The INSIG-1 is highly expressed in the adult liver and plays a role in sterol homeostasis. INSIG-1 was downregulated in 25-HC-treated HCV replicon cells compared with HCV replicon-harbouring cells (Table ). INSIG-1 represents a class of genes that are negatively regulated during HCV replication under metabolic conditions that are induced by the inhibitor of the mevalonate pathway, 25-HC. This transcript is also upregulated in an outcome-specific manner in the chimpanzee who experienced a clearance episode (SC and TC) but not in that which resulted in a persistent (PS) infection (Table ).
The NK4 gene was chosen for further validation because its expression seems to be correlated with a clearance state in the chimpanzee and it was found to be upregulated in the 25-HC-treated HCV replicon cells compared with naïve Huh-7 cells treated with 25-HC (Table ). Unlike PROX1 and INSIG1, NK4 represents a class of genes that are induced upon HCV replication and appears to be modulated by treatment with 25-HC.
Finally, the UBD gene was chosen for further validation because its expression was increased dramatically in the chimpanzees associated with sustained and transient clearance, 43-fold and 7-fold, respectively, and it was upregulated in 25-HC-treated HCV replicon cells compared with naïve Huh-7 cells treated with 25-HC (Table ). UBD represents a class of genes that are induced during HCV replication when an antiviral state exists within the host cell, as demonstrated by the increase in expression in HCV replicon harbouring cells treated with 25-HC. This gene may serve as a prognostic marker for HCV infection.
Next we examined the overall gene expression patterns for these four genes (Table ) in the TC, SC, and PS chimpanzees. The expression levels of the four genes in infected chimpanzees were plotted as a function of time alongside the HCV RNA levels measured in the chimpanzees [11
] (Fig. ). The comparative analysis between the chimpanzee and replicon data sets was done based on the first three chimpanzee time points, which corresponded to the initial rise in viremia [11
]. However, the expression of the four genes of interest was determined and plotted for the entire measured time course of infection for the chimpanzees in Fig. . In general, the gene expression levels of the four genes paralleled the HCV levels in the chimpanzees that displayed sustained clearance (SC) and transient clearance (TC) of HCV. The overall gene expression levels were lowest in the persistently infected (PS) chimpanzee compared to the SC and TC chimpanzees. Also, the overall gene expression levels were higher for all four genes in the TC chimpanzee, which also had the highest viral titres (Fig. ).
Figure 3 Inhibition of HCV RNA replication by 25-hydroxycholesterol in a dose-dependent manner. Activity data from Huh-7 cells transiently transfected with luc/NS3-3'/5.1 replicon RNA and treated with 25-HC or 100 U/ml IFNγ for 24 h. Bars represent the (more ...)
Expression of PROX1 in the HCV-infected chimpanzees early post-infection decreased slightly within the first six days for the SC chimpanzee, increased within the first eight days in the TC chimpanzee, and decreased within the first eight days in the PS chimpanzee (Fig. ). Throughout the remaining course of infection, PROX1 levels roughly correlated with HCV levels in the SC and TC chimpanzees, while in the PS chimpanzee the PROX1 levels remained lower than the HCV levels. The expression level of INSIG-1 early post-infection increased in the SC and TC chimpanzees while it varied only slightly within the first eighteen days post-infection in the PS chimpanzee (Fig. ). Thereafter, the INSIG-1 levels closely paralleled the HCV RNA levels in the SC and TC chimpanzees and rose to meet the HCV level in the PS chimpanzee. The NK4 levels during the initial rise in viremia in the chimpanzees paralleled the rising HCV levels in the SC and TC chimpanzees (Fig. ). In contrast, in the PS chimpanzee the NK4 levels decreased within the first eight days of infection and then increased along with the rising HCV RNA level. Finally, the UBD pattern of expression basically paralleled the levels of HCV RNA in the SC and TC chimpanzees with some variation in the absolute levels of expression relative to that of the HCV RNA (Fig. ). In the PS chimpanzee, the UBD expression level remained close to baseline levels in the first eighteen days post-infection, and then the UBD expression steadily increased during the remaining time course of HCV infection.
To ascertain the importance of the four genes for HCV replication, siRNA knockdown and overexpression studies were carried out in Huh-7 cells harbouring HCV replicon RNA. RT-PCR and northern blot analyses were used to confirm siRNA and overexpression vector function in each case (data not shown). Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 HCV replicons [8
] were treated with siRNAs against the four genes in the absence or presence of 25-HC (Fig. ). As controls, the HCV replicon cells were also treated with GL2 and GL3 siRNAs, a positive control that targets the luciferase gene expressed from the HCV replicon RNA and a negative control, respectively, in addition to IFNγ treatment [15
]. Interestingly, although the PROX1 siRNA did not affect HCV replication on its own, it enhanced the inhibitory effect of treatment with 5 μM 25-HC when compared to mock-transfected replicon cells treated with 5 μM 25-HC (p
< 0.01) as indicated (Fig. ). Overexpression of PROX1 in replicon cells did not affect HCV replication (Fig. ). Knockdown of INSIG-1 with homologous siRNAs did not affect HCV replication in replicon cells either in the absence or presence of 25-HC (Fig. ). Similarly, overexpression of INSIG-1 in replicon cells did not affect HCV replication (Fig. ). Although the transcriptional profiling showed that NK4 was upregulated in replicon cells treated with 25-HC, siRNA knockdown of NK4 did not affect HCV replication (Fig. ). However, overexpression of NK4 affected HCV replication in the presence of 25-HC treatment, causing a significant decrease in HCV replication compared to replicon cells that were transfected with the backbone β-gal control vector and treated with 25-HC (p
< 0.05; Fig. ). From the profiling data, UBD was upregulated in 25-HC-treated replicon cells compared with naïve Huh-7 cells treated with 25-HC. However, neither knockdown of UBD using siRNA duplexes (Fig. ) nor overexpression of UBD (Fig. ) had an affect on HCV replication.
Figure 5 The effect of 25-hydroxycholesterol and siRNA knockdown or gene overexpression on HCV RNA replication and translation. Huh-7 cells stably expressing neo/luc/NS3-3'/5.1 or neo/NS3-3'/5.1 subgenomic replicons were transiently transfected with siRNA duplexes (more ...)
To determine if siRNA treatment or overexpression had any affect on the expression of the HCV nonstructural proteins NS3 and NS5A, Huh-7 cells stably expressing neo/NS3-3'/5.1 replicons were transiently transfected with siRNA or overexpression constructs and western blot analyses were conducted. As expected, siRNA knockdown of PROX1 gene expression resulted in complete loss of PROX1 protein expression (Fig. ). However, knockdown of PROX1 expression did not have any affect on NS3 or NS5A protein levels (Fig. ). Corresponding to their negative effect on HCV replication, IFNγ and 25-HC treatment of replicon cells caused a decrease in the levels of NS3 and NS5A, with the effect being more pronounced for NS5A as we had previously reported [25
]. Interestingly, overexpression of INSIG-1 caused an increase in the levels of both HCV non-structural proteins (Fig. ). Overexpression of NK4 and UBD had no effect on the levels of HCV non-structural proteins (Fig. ).