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Hepatitis C virus infection activates an innate pathway involving IKK-α in lipogenesis and viral assembly.

Li Q, Pène V, Krishnamurthy S, Cha H, Liang TJ - Nat. Med. (2013)

Bottom Line: Here we describe a new nuclear factor κB (NF-κB)-independent and kinase-mediated nuclear function of IKK-α in HCV assembly.HCV, through its 3' untranslated region, interacts with DEAD box polypeptide 3, X-linked (DDX3X) to activate IKK-α, which translocates to the nucleus and induces a CBP/p300-mediated transcriptional program involving sterol regulatory element-binding proteins (SREBPs).This innate pathway induces lipogenic genes and enhances core-associated lipid droplet formation to facilitate viral assembly.

View Article: PubMed Central - PubMed

Affiliation: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Hepatitis C virus (HCV) interacts extensively with host factors to not only establish productive infection but also trigger unique pathological processes. Our recent genome-wide siRNA screen demonstrated that IκB kinase-α (IKK-α) is a crucial host factor for HCV. Here we describe a new nuclear factor κB (NF-κB)-independent and kinase-mediated nuclear function of IKK-α in HCV assembly. HCV, through its 3' untranslated region, interacts with DEAD box polypeptide 3, X-linked (DDX3X) to activate IKK-α, which translocates to the nucleus and induces a CBP/p300-mediated transcriptional program involving sterol regulatory element-binding proteins (SREBPs). This innate pathway induces lipogenic genes and enhances core-associated lipid droplet formation to facilitate viral assembly. Chemical inhibitors of IKK-α suppress HCV infection and IKK-α-induced lipogenesis, offering a proof-of-concept approach for new HCV therapeutic development. Our results show that HCV uses a novel mechanism to exploit intrinsic innate responses and hijack lipid metabolism, which may contribute to high chronicity rates and the pathological hallmark of steatosis in HCV infection.

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IKKα function in HCV assembly and HCV-induced LD formation. (a) Effects of various siRNAs on HCV JFH-1/P7-Luc RNA replication in CD81-deficient Huh7.25 cells. (b) Effects of various siRNAs on HCV subgenomic replicon assay. (a,b) Values were normalized as relative units to siNT control, and error bars represent ± s.d. of quintuplicate experiments. (c) LD contents (BODIPY) and HCV core expression in Huh7.5.1 cells treated with siNT or siIKKα. LD number: mean of >150 cells ± s.d. Percent of LD-positive area: mean of >150 cells ± s.d. LD mean fluorescence intensity: mean of >300 cells ± s.d. (d) Huh7.5.1 cells were transfected with control, HA-IKKα WT or HA-IKKα KM plasmid and then stained for HA-tagged IKKα expression and LD contents. LD numbers, positive area and mean fluorescence intensity were quantified: mean of >30 cells per condition ± s.d. (e) Effect of IKKα silencing on HCV 3’UTR-mediated elevation of LD contents in Huh7.5.1 cells. For all microscopic images, scale bars represent 20 µm. **, P < 0.01. NS, not significant.
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Figure 2: IKKα function in HCV assembly and HCV-induced LD formation. (a) Effects of various siRNAs on HCV JFH-1/P7-Luc RNA replication in CD81-deficient Huh7.25 cells. (b) Effects of various siRNAs on HCV subgenomic replicon assay. (a,b) Values were normalized as relative units to siNT control, and error bars represent ± s.d. of quintuplicate experiments. (c) LD contents (BODIPY) and HCV core expression in Huh7.5.1 cells treated with siNT or siIKKα. LD number: mean of >150 cells ± s.d. Percent of LD-positive area: mean of >150 cells ± s.d. LD mean fluorescence intensity: mean of >300 cells ± s.d. (d) Huh7.5.1 cells were transfected with control, HA-IKKα WT or HA-IKKα KM plasmid and then stained for HA-tagged IKKα expression and LD contents. LD numbers, positive area and mean fluorescence intensity were quantified: mean of >30 cells per condition ± s.d. (e) Effect of IKKα silencing on HCV 3’UTR-mediated elevation of LD contents in Huh7.5.1 cells. For all microscopic images, scale bars represent 20 µm. **, P < 0.01. NS, not significant.

Mentions: To investigate the step of HCV life cycle where IKKα is required, we applied multiple virologic assays. IKKα silencing preferentially affected extracellular HCV RNA levels in HCVcc infection system (Fig. 1b). We therefore specifically examined single-cycle replication by transfecting genomic HCV RNA into CD81-deficient Huh7 cells (Huh7.25)21 and showed that HCV replication was not affected by IKKα silencing (Fig. 2a). IKKα silencing had no effect on assays targeting individual steps of HCV life cycle including entry (HCV pseudovirus assay), translation (HCV IRES-driven reporter), and replication (subgenomic replicon) (Supplementary Fig. 3 and Fig. 2b), consistent with a predominant role of IKKα in the late stage of viral life cycle.


Hepatitis C virus infection activates an innate pathway involving IKK-α in lipogenesis and viral assembly.

Li Q, Pène V, Krishnamurthy S, Cha H, Liang TJ - Nat. Med. (2013)

IKKα function in HCV assembly and HCV-induced LD formation. (a) Effects of various siRNAs on HCV JFH-1/P7-Luc RNA replication in CD81-deficient Huh7.25 cells. (b) Effects of various siRNAs on HCV subgenomic replicon assay. (a,b) Values were normalized as relative units to siNT control, and error bars represent ± s.d. of quintuplicate experiments. (c) LD contents (BODIPY) and HCV core expression in Huh7.5.1 cells treated with siNT or siIKKα. LD number: mean of >150 cells ± s.d. Percent of LD-positive area: mean of >150 cells ± s.d. LD mean fluorescence intensity: mean of >300 cells ± s.d. (d) Huh7.5.1 cells were transfected with control, HA-IKKα WT or HA-IKKα KM plasmid and then stained for HA-tagged IKKα expression and LD contents. LD numbers, positive area and mean fluorescence intensity were quantified: mean of >30 cells per condition ± s.d. (e) Effect of IKKα silencing on HCV 3’UTR-mediated elevation of LD contents in Huh7.5.1 cells. For all microscopic images, scale bars represent 20 µm. **, P < 0.01. NS, not significant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3676727&req=5

Figure 2: IKKα function in HCV assembly and HCV-induced LD formation. (a) Effects of various siRNAs on HCV JFH-1/P7-Luc RNA replication in CD81-deficient Huh7.25 cells. (b) Effects of various siRNAs on HCV subgenomic replicon assay. (a,b) Values were normalized as relative units to siNT control, and error bars represent ± s.d. of quintuplicate experiments. (c) LD contents (BODIPY) and HCV core expression in Huh7.5.1 cells treated with siNT or siIKKα. LD number: mean of >150 cells ± s.d. Percent of LD-positive area: mean of >150 cells ± s.d. LD mean fluorescence intensity: mean of >300 cells ± s.d. (d) Huh7.5.1 cells were transfected with control, HA-IKKα WT or HA-IKKα KM plasmid and then stained for HA-tagged IKKα expression and LD contents. LD numbers, positive area and mean fluorescence intensity were quantified: mean of >30 cells per condition ± s.d. (e) Effect of IKKα silencing on HCV 3’UTR-mediated elevation of LD contents in Huh7.5.1 cells. For all microscopic images, scale bars represent 20 µm. **, P < 0.01. NS, not significant.
Mentions: To investigate the step of HCV life cycle where IKKα is required, we applied multiple virologic assays. IKKα silencing preferentially affected extracellular HCV RNA levels in HCVcc infection system (Fig. 1b). We therefore specifically examined single-cycle replication by transfecting genomic HCV RNA into CD81-deficient Huh7 cells (Huh7.25)21 and showed that HCV replication was not affected by IKKα silencing (Fig. 2a). IKKα silencing had no effect on assays targeting individual steps of HCV life cycle including entry (HCV pseudovirus assay), translation (HCV IRES-driven reporter), and replication (subgenomic replicon) (Supplementary Fig. 3 and Fig. 2b), consistent with a predominant role of IKKα in the late stage of viral life cycle.

Bottom Line: Here we describe a new nuclear factor κB (NF-κB)-independent and kinase-mediated nuclear function of IKK-α in HCV assembly.HCV, through its 3' untranslated region, interacts with DEAD box polypeptide 3, X-linked (DDX3X) to activate IKK-α, which translocates to the nucleus and induces a CBP/p300-mediated transcriptional program involving sterol regulatory element-binding proteins (SREBPs).This innate pathway induces lipogenic genes and enhances core-associated lipid droplet formation to facilitate viral assembly.

View Article: PubMed Central - PubMed

Affiliation: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Hepatitis C virus (HCV) interacts extensively with host factors to not only establish productive infection but also trigger unique pathological processes. Our recent genome-wide siRNA screen demonstrated that IκB kinase-α (IKK-α) is a crucial host factor for HCV. Here we describe a new nuclear factor κB (NF-κB)-independent and kinase-mediated nuclear function of IKK-α in HCV assembly. HCV, through its 3' untranslated region, interacts with DEAD box polypeptide 3, X-linked (DDX3X) to activate IKK-α, which translocates to the nucleus and induces a CBP/p300-mediated transcriptional program involving sterol regulatory element-binding proteins (SREBPs). This innate pathway induces lipogenic genes and enhances core-associated lipid droplet formation to facilitate viral assembly. Chemical inhibitors of IKK-α suppress HCV infection and IKK-α-induced lipogenesis, offering a proof-of-concept approach for new HCV therapeutic development. Our results show that HCV uses a novel mechanism to exploit intrinsic innate responses and hijack lipid metabolism, which may contribute to high chronicity rates and the pathological hallmark of steatosis in HCV infection.

Show MeSH
Related in: MedlinePlus