Hepatitis C virus NS4B targets lipid droplets through hydrophobic residues in the amphipathic helices

Tanaka, Torahiko; Kuroda, Kensuke; Ikeda, Masanori; Wakita, Takaji; Kato, Naoya; Makishima, Makoto

doi:10.1194/jlr.m026443

Cited by 24 publications

(22 citation statements)

References 51 publications

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“…HCV core protein recruits HCV RNA, nonstructural proteins, and replication complexes to LD-associated membranes, and this recruitment is critical for infectious virus particle production (29). HCV NS4B is known to play a crucial role in virus replication at the site of virion formation, namely, the microenvironment associated with LDs (35). Mutations of HCV core and NS5A result in a failure to associate with LDs and impair the production of infectious virus (26), indicating the importance of these proteins in HCV replication and assembly.…”

Section: Discussionmentioning

confidence: 99%

Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus

Bose

Kim

Meyer

et al. 2014

J Virol

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We have previously shown that hepatitis C virus (HCV) infection modulates the expression of forkhead box transcription factors, including FoxO1 and FoxA2, which play key roles in gluconeogenesis and ␤-oxidation of fatty acid, respectively. The aim of the present study was to determine the role of forkhead box transcription factors in modulating lipid metabolism. HCV infection or core protein expression alone in transfected Huh7. H epatitis C virus (HCV)is an important cause of morbidity and mortality worldwide, causing a spectrum of disease ranging from an asymptomatic carrier state to end-stage liver disease (1-4). The most important feature of HCV infection is the development of chronic hepatitis in a significant number of infected individuals and the potential for disease progression to metabolic disorders, fibrosis/cirrhosis, and hepatocellular carcinoma (1-3, 5). We have previously shown that HCV modulates signaling pathways in inducing insulin resistance (6, 7).Insulin regulates the expression of key enzymes involved in glucose and lipid metabolism by modulating the activity of specific forkhead box transcription factors (FoxO1 and FoxA2) in the liver. FoxO1 mediates the expression of genes involved in both glucose and lipid metabolism in the liver (8, 9). Insulin suppresses the expression of key gluconeogenic enzymes, including glucose 6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PCK2), by stimulating the translocation of FoxO1 outside the nucleus (10). Increased glucose production can activate genes involved in lipid metabolism, including sterol regulatory element binding protein 1c (SREBP-1c) and fatty acid synthase (FASN).On the other hand, FoxA2 controls hepatic lipid metabolism in type 2 diabetes, improving insulin resistance (11,12). Thus, an interruption in insulin signaling may affect metabolic regulation. During chronic HCV infection, insulin resistance may elicit a vicious cycle for manipulating the functions of FoxA2, preventing optimal stimulation of normal metabolic functions of the liver. Lipid homeostasis requires balancing metabolic vectors, including lipogenesis, export, and degradation (␤-oxidation), a significant component of which is orchestrated by a family of membrane-bound master regulator transcription factors designated sterol regulatory element binding proteins (SREBPs) that regulate the expression of more than 30 genes involved in the synthesis and uptake of cholesterol, fatty acids, triglycerides, and phospholipids

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Section: Discussionmentioning

confidence: 99%

Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus

Bose

Kim

Meyer

et al. 2014

J Virol

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“…1E). In fact, recent studies have emphasized the association of lipid droplets and HCV RNA replication (43). Further research is required to clarify the manner by which HCV RNA replication is influenced by intracellular fatty acid homeostasis.…”

Section: Figmentioning

confidence: 99%

Hepatitis C Virus Entry Is Impaired by Claudin-1 Downregulation in Diacylglycerol Acyltransferase-1-Deficient Cells

Sung

Murayama

Kang

et al. 2014

J Virol

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Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4␣ (HNF4␣) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low-dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV. IMPORTANCEIn this study, we report the novel effect of complete silencing of DGAT1 on the entry of HCV. DGAT1 was recently reported as a host factor of HCV, involved in the assembly of HCV by facilitating the trafficking of the HCV core protein to lipid droplets. We achieved complete and long-term silencing of DGAT1 by either TALEN or repeated transduction of lentivirus shRNA. We found that HCV entry was severely impaired in DGAT1-silenced cell lines. The impairment of HCV entry was caused by CLDN1 downregulation, and the expression of HNF4␣ and other hepatocyte-specific genes was also downregulated in DGAT1-silenced cell lines. Our results suggest new roles of DGAT1 in human liver-derived cells: maintaining intracellular lipid homeostasis and affecting HCV entry by modulating CLDN1 expression. H epatitis C virus (HCV) infection poses a major threat to human health, with a prevalence of more than 160 million people worldwide (1). In addition to a combination regimen of pegylated interferon alpha (IFN-␣) and ribavirin, drugs acting directly on HCV have now been developed, although these direct-acting antivirals may prompt the emergence of resistant strains (2, 3). Our increasing understanding of the HCV-host interactions is allowing novel therapeutic approaches to be developed that modulate host factors that are required for viral entry, replication, and egress; these factors may have a higher genetic barrier to viral resistance (4).Diacylglycerol acyltransferase-1 (DGAT1) is one of two known DGAT enzymes catalyzing the final step in triglyceride biosynthesis (5, 6). The expression of DGAT1 and its physiologic role differ in humans and mice. In mice, DGAT1 is expressed in many organs, including the skeletal muscle, heart, and intestines, but it is bar...

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“…In this environment, NS4B protein likely provides a platform for interactions of proteins that comprise the HCV replication complex, as supported by evidence of genetic and physical interactions with other proteins in the complex (15)(16)(17)(18)(19). Several biochemical functions of NS4B have been described, including protein multimerization (20)(21)(22), ATPase/GTPase activity (23,24), RNA binding (25), and interaction with membranes or lipid droplets (26)(27)(28). Previous reports have identified small-molecule binders of NS4B that are associated with inhibition of particular biochemical functions.…”

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confidence: 99%

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

Pouliot

Thomson

Xie

et al. 2015

Antimicrob Agents Chemother

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dThe hepatitis C virus (HCV) NS4B protein is an antiviral therapeutic target for which small-molecule inhibitors have not been shown to exhibit in vivo efficacy. We describe here the in vitro and in vivo antiviral activity of GSK8853, an imidazo[1,2-a]pyrimidine inhibitor that binds NS4B protein. GSK8853 was active against multiple HCV genotypes and developed in vitro resistance mutations in both genotype 1a and genotype 1b replicons localized to the region of NS4B encoding amino acids 94 to 105. A 20-day in vitro treatment of replicons with GSK8853 resulted in a 2-log drop in replicon RNA levels, with no resistance mutation breakthrough. Chimeric replicons containing NS4B sequences matching known virus isolates showed similar responses to a compound with genotype 1a sequences but altered efficacy with genotype 1b sequences, likely corresponding to the presence of known resistance polymorphs in those isolates. In vivo efficacy was tested in a humanized-mouse model of HCV infection, and the results showed a 3-log drop in viral RNA loads over a 7-day period. Analysis of the virus remaining at the end of in vivo treatment revealed resistance mutations encoding amino acid changes that had not been identified by in vitro studies, including NS4B N56I and N99H. Our findings provide an in vivo proof of concept for HCV inhibitors targeting NS4B and demonstrate both the promise and potential pitfalls of developing NS4B inhibitors. Hepatitis C virus (HCV) is a significant public health threat, with up to 3% of the world population estimated to be harboring the infection. Although the virus can be naturally cleared, it more often becomes established as a chronic infection with an increased risk of poor long-term outcomes, including liver cirrhosis and cancer (1). Treatment of the disease historically used a combination of ribavirin and pegylated interferon, a cocktail with a 50% cure rate against genotype 1 but with a high likelihood of undesirable side effects (2, 3). The regulatory approval of directacting small-molecule antivirals represents a major advance in therapeutics by increasing the chance of efficacious treatment (4, 5), but there is still a need for novel antiviral therapies with reduced side effects and complementary resistance profiles. HCV inhibitors of the NS3 protease, of the multifunctional protein NS5A, and of the NS5B RNA-dependent RNA polymerase have been shown to be efficacious in the clinic, but inhibitors of NS4B have not been validated in vivo. We (6-8) and others (9-12) have described compounds targeting NS4B in vitro that, if developed, would offer a complementary mechanism that may improve the standard of care for hepatitis C virus infection treatment.HCV RNA replication occurs in vesicle-induced intracellular membranes derived from the cellular endoplasmic reticulum (ER), catalyzed by a complex of proteins encoded by the virus and host factors. NS4B is an integral membrane protein (13) that induces the rearrangement of intracellular membranes into a "membranous web," a collection of vesicles th...

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Hepatitis C virus NS4B targets lipid droplets through hydrophobic residues in the amphipathic helices

Cited by 24 publications

References 51 publications

Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus

Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus

Hepatitis C Virus Entry Is Impaired by Claudin-1 Downregulation in Diacylglycerol Acyltransferase-1-Deficient Cells

Preclinical Characterization and In Vivo Efficacy of GSK8853, a Small-Molecule Inhibitor of the Hepatitis C Virus NS4B Protein

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