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Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus.

Pezacki JP, Sagan SM, Tonary AM, Rouleau Y, Bélanger S, Supekova L, Su AI - BMC Chem Biol (2009)

Bottom Line: These data were compared with gene expression profiles from HCV-infected chimpanzees.Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell.These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

View Article: PubMed Central - HTML - PubMed

Affiliation: Steacie Institute for Molecular Sciences, The National Research Council of Canada, Ottawa, K1A 0R6 Canada . John.pezacki@nrc.ca

ABSTRACT

Background: Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees.

Results: Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol.

Conclusion: Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

No MeSH data available.


Related in: MedlinePlus

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 mean ± standard deviation of triplicate samples. An asterisk above a bar indicates a significant decrease in replication compared to untreated cells (p < 0.01).
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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 mean ± standard deviation of triplicate samples. An asterisk above a bar indicates a significant decrease in replication compared to untreated cells (p < 0.01).

Mentions: Next we examined the overall gene expression patterns for these four genes (Table 4) 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. 3). 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. 4. 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. 4).


Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus.

Pezacki JP, Sagan SM, Tonary AM, Rouleau Y, Bélanger S, Supekova L, Su AI - BMC Chem Biol (2009)

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 mean ± standard deviation of triplicate samples. An asterisk above a bar indicates a significant decrease in replication compared to untreated cells (p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651120&req=5

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 mean ± standard deviation of triplicate samples. An asterisk above a bar indicates a significant decrease in replication compared to untreated cells (p < 0.01).
Mentions: Next we examined the overall gene expression patterns for these four genes (Table 4) 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. 3). 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. 4. 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. 4).

Bottom Line: These data were compared with gene expression profiles from HCV-infected chimpanzees.Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell.These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

View Article: PubMed Central - HTML - PubMed

Affiliation: Steacie Institute for Molecular Sciences, The National Research Council of Canada, Ottawa, K1A 0R6 Canada . John.pezacki@nrc.ca

ABSTRACT

Background: Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees.

Results: Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol.

Conclusion: Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

No MeSH data available.


Related in: MedlinePlus