Limits...
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

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 different outcomes of infection (SC, TC, and PS) [11]. Green lines represent log HCV RNA patterns, with values below the threshold for detection represented by zero.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651120&req=5

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 different outcomes of infection (SC, TC, and PS) [11]. Green lines represent log HCV RNA patterns, with values below the threshold for detection represented by zero.

Mentions: 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-I389neo/NS3-3'/5.1 or replicon RNA from pFK-I389neo/NS3-3'/Δ5B replicon RNAs (Fig. 2a); and (4) Huh-7 cells transiently transfected with pFK-I389neo/NS3-3'/5.1 or pFK-I389neo/NS3-3'/Δ5B replicon RNAs and treated with 25-HC as indicated in Fig. 2b. The experiments were performed analogously to previous studies of antiviral small molecules on HCV replication [25-27]. As previously reported [8,11], 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-I389luc/NS3-3'/5.1 (Fig. 2a).


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)

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 different outcomes of infection (SC, TC, and PS) [11]. Green lines represent log HCV RNA patterns, with values below the threshold for detection represented by zero.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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 different outcomes of infection (SC, TC, and PS) [11]. Green lines represent log HCV RNA patterns, with values below the threshold for detection represented by zero.
Mentions: 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-I389neo/NS3-3'/5.1 or replicon RNA from pFK-I389neo/NS3-3'/Δ5B replicon RNAs (Fig. 2a); and (4) Huh-7 cells transiently transfected with pFK-I389neo/NS3-3'/5.1 or pFK-I389neo/NS3-3'/Δ5B replicon RNAs and treated with 25-HC as indicated in Fig. 2b. The experiments were performed analogously to previous studies of antiviral small molecules on HCV replication [25-27]. As previously reported [8,11], 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-I389luc/NS3-3'/5.1 (Fig. 2a).

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