Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture

Harak, Christian; Meyrath, Max; Romero-Brey, Inés; Schenk, Christian; Gondeau, Claire; Schult, Philipp; Esser-Nobis, Katharina; Saeed, Mohsan; Neddermann, Petra; Schnitzler, Paul; Gotthardt, Daniel; Pérez‐del‐Pulgar, Sofía; Neumann‐Haefelin, Christoph; Thimme, Robert; Meuleman, Philip; Vondran, Florian W. R.; Francesco, Raffaele De; Rice, Charles M.; Bartenschlager, Ralf; Lohmann, Volker

doi:10.1038/nmicrobiol.2016.247

Cited by 56 publications

(102 citation statements)

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“…Saeed et al recently demonstrated that SEC14L2 expression in hepatoma cell lines promotes HCV RNA replication following inoculation with patient sera, through enhancement of vitamin E–mediated protection against lipid peroxidation . In addition, Harak et al elegantly showed that inhibition of phosphatidylinositol 4‐kinase alpha in HuH‐7 cells allows efficient replication of patient‐derived viruses . In 2013, Steenbergen et al demonstrated that the production of HCVcc was improved when using HuH‐7 cells that had been differentiated by supplementing tissue culture medium with human serum for 7 days .…”

Section: Discussionmentioning

confidence: 99%

“…(30) In addition, Harak et al elegantly showed that inhibition of phosphatidylinositol 4-kinase alpha in HuH-7 cells allows efficient replication of patient-derived viruses. (31) In 2013, Steenbergen et al demonstrated that the production of HCVcc was improved when using HuH-7 cells that had been differentiated by supplementing tissue culture medium with human serum for 7 days. (32) In these conditions, cells undergo growth arrest, have a hepatocytelike morphology, and increase the expression of hepatocyte differentiation markers, which enables the production of large amounts of virus that more closely resemble HCV present in the serum of infected patients.…”

Section: Discussionmentioning

confidence: 99%

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Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles

et al. 2017

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The development of different cell culture models has greatly contributed to increased understanding of the hepatitis C virus (HCV) life cycle. However, it is still challenging to grow HCV clinical isolates in cell culture. If overcome, this would open new perspectives to study HCV biology, including drug‐resistant variants emerging with new antiviral therapies. In this study we hypothesized that this hurdle could be due to the presence of inhibitory factors in patient serum. Combining polyethylene glycol precipitation, iodixanol gradient, and size‐exclusion chromatography, we obtained from HCV‐seronegative sera a purified fraction enriched in inhibitory factors. Mass spectrometric analysis identified apolipoprotein(a) (apo[a]) as a potential inhibitor of HCV entry. Apo(a) consists of 10 kringle IV domains (KIVs), one kringle V domain, and an inactive protease domain. The 10 KIVs are present in a single copy with the exception of KIV type 2 (KIV2), which is encoded in a variable number of tandemly repeated copies, giving rise to numerous apo(a) size isoforms. In addition, apo(a) covalently links to the apolipoprotein B component of a low‐density lipoprotein through a disulfide bridge to form lipoprotein(a). Using a recombinant virus derived from the JFH1 strain, we confirmed that plasma‐derived and recombinant lipoprotein(a) as well as purified recombinant apo(a) variants were able to specifically inhibit HCV by interacting with infectious particles. Our results also suggest that small isoforms are less inhibitory than the large ones. Finally, we observed that the lipoprotein moiety of HCV lipoviroparticles was essential for inhibition, whereas functional lysine‐binding sites in KIV7, KIV8, and KIV10 were not required. Conclusions: Our results identify apo(a) as an additional component of the lipid metabolism modulating HCV infection. (Hepatology 2017;65:1851‐1864)

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles

et al. 2017

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“…Regarding the critical involvement of finely tuned PI4KIIIα activation in HCV replication (32), our study provides novel insight into the modulatory role of hCKα during HCV replication. hCKα functions as an indispensable, upstream coordinator of PI4KIIIα and NS5A by bridging them, reshaping their functions, and retargeting their intracellular localization to the PI4P-enriched, ER-derived membrane platform (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, Harak et al demonstrated that HCV depends on optimal levels of PI4KIIIα activity for proper replication and showed that the replication of various genotypes requires PI4KIIIα activation at a low PI4KIIIα protein level, such as that observed for primary hepatocytes (32). Nevertheless, in cultured hepatoma Huh7 cells, which already express higher levels of PI4KIIIα than primary hepatocytes, NS5A-induced PI4KIIIα activation leads to excess PI4P production that is harmful to their replication (32).…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, in cultured hepatoma Huh7 cells, which already express higher levels of PI4KIIIα than primary hepatocytes, NS5A-induced PI4KIIIα activation leads to excess PI4P production that is harmful to their replication (32). To manage the growth disadvantage in Huh7 cells, these isolates have evolved a mechanism through adaptive mutations, such as S2204R in NS5A and R2884G in NS5B, to limit excess PI4KIIIα activation, thus ensuring their proper replication in Huh7 cells (32). Strikingly, the genotype 2a JFH1 virus is an exception, because it does not require adaptive mutations for robust replication in Huh7 cells (33).…”

Section: Introductionmentioning

confidence: 99%

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Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication

Wong

Chen

2017

J Virol

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In this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication.IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the “membranous web” structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

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Tracking Hepatitis C Virus Interactions with the Hepatic Lipid Metabolism

Vièyres

Pietschmann

2020

The Liver

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Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture

Cited by 56 publications

References 50 publications

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles

Apolipoprotein(a) inhibits hepatitis C virus entry through interaction with infectious particles

Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication

Tracking Hepatitis C Virus Interactions with the Hepatic Lipid Metabolism

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