[Show abstract][Hide abstract]ABSTRACT: It is well known that occludin (OCLN) is involved in hepatitis C virus (HCV) entry into hepatocytes, but there has been no conclusive evidence that OCLN is essential for HCV infection. In this study, we first established an OCLN-knockout cell line derived from human hepatic Huh7.5.1-8 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, in which two independent targeting plasmids expressing single-guide RNAs were used. One established cell clone, named OKH-4, had the OCLN gene truncated in the N-terminal region, and a complete defect of the OCLN protein was shown using immunoblot analysis. Infection of OKH-4 cells with various genotypes of HCV was abolished, and exogenous expression of the OCLN protein in OKH-4 cells completely reversed permissiveness to HCV infection. In addition, using a co-culture system of HCV-infected Huh7.5.1-8 cells with OKH-4 cells, we showed that OCLN is also critical for cell-to-cell HCV transmission. Thus, we concluded that OCLN is essential for HCV infection of human hepatic cells. Further experiments using HCV genomic RNA-transfected OKH-4 cells or HCV subgenomic replicon-harboring OKH-4 cells suggested that OCLN is mainly involved in the entry step of the HCV life cycle. It was also demonstrated that the second extracellular loop of OCLN, especially the two cysteine residues, is critical for HCV infection of hepatic cells. OKH-4 cells may be a useful tool for understanding not only the entire mechanism of HCV entry, but also the biological functions of OCLN.
[Show abstract][Hide abstract]ABSTRACT: Based on multipurpose cohort studies, coffee consumption reduces the risk of hepatocellular carcinoma, one of the main causes of which is hepatitis C virus (HCV) infection. Here, we focused on the effect of caffeic acid, a major organic acid derived from coffee, on the propagation of HCV using an in vitro naïve HCV particle-infection and production system within human hepatoma-derived Huh7.5.1-8 cells. When cells were treated with 1% coffee extract or 0.1% caffeic acid for 1-h after HCV infection, the amount of HCV particles released into the medium at 3 and 4 days post-infection were considerably decreased. HCV-infected cells were cultured with 0.001% caffeic acid for 4 days, which was sufficient to decrease the amount of HCV particles released into the medium. Caffeic acid treatment inhibited the initial stage of HCV infection, i.e., between virion entry and the translation of the RNA genome. This inhibitory effect was observed against both HCV genotypes 1b and 2a. These results suggested that treatment of cells with caffeic acid inhibited HCV propagation.
Preview · Article · Jan 2015 · Japanese journal of infectious diseases
[Show abstract][Hide abstract]ABSTRACT: Unlabelled:
Hepatitis C virus (HCV) exploits host membrane cholesterol and its metabolism for progeny virus production. Here, we examined the impact of targeting cellular squalene synthase (SQS), the first committed enzyme for cholesterol biosynthesis, on HCV production. By using the HCV JFH-1 strain and human hepatoma Huh-7.5.1-derived cells, we found that the SQS inhibitors YM-53601 and zaragozic acid A decreased viral RNA, protein, and progeny production in HCV-infected cells without affecting cell viability. Similarly, small interfering RNA (siRNA)-mediated knockdown of SQS led to significantly reduced HCV production, confirming the enzyme as an antiviral target. A metabolic labeling study demonstrated that YM-53601 suppressed the biosynthesis of cholesterol and cholesteryl esters at antiviral concentrations. Unlike YM-53601, the cholesterol esterification inhibitor Sandoz 58-035 did not exhibit an antiviral effect, suggesting that biosynthesis of cholesterol is more important than that of cholesteryl esters for HCV production. YM-53601 inhibited transient replication of a JFH-1 subgenomic replicon and entry of JFH-1 pseudoparticles, suggesting that at least suppression of viral RNA replication and entry contributes to the antiviral effect of the drug. Collectively, our findings highlight the importance of the cholesterol biosynthetic pathway in HCV production and implicate SQS as a potential target for antiviral strategies against HCV.
Hepatitis C virus (HCV) is known to be closely associated with host cholesterol and its metabolism throughout the viral life cycle. However, the impact of targeting cholesterol biosynthetic enzymes on HCV production is not fully understood. We found that squalene synthase, the first committed enzyme for cholesterol biosynthesis, is important for HCV production, and we propose this enzyme as a potential anti-HCV target. We provide evidence that synthesis of free cholesterol is more important than that of esterified cholesterol for HCV production, highlighting a marked free cholesterol dependency of HCV production. Our findings also offer a new insight into a role of the intracellular cholesterol pool that is coupled to its biosynthesis in the HCV life cycle.
Preview · Article · Dec 2014 · Journal of Virology
[Show abstract][Hide abstract]ABSTRACT: An efficient cell culture and infection system for hepatitis C virus (HCV) is useful for analyzing the complete virus life cycle. Human hepatic Huh7.5.1 cells and a HCV-JFH1 strain have been widely employed for infection experiments. Huh7.5.1 cells that we have cultured exhibited heterogeneous phenotypes of HCV infection. By single-cell cloning of Huh7.5.1 cells, we isolated a clone highly permissive to HCV and a CD81-defective clone nonpermissive to HCV, designated as Huh7.5.1-8 and Huh7.5.1-5, respectively. Expression of CD81 into Huh7.5.1-5 cells restored the permissiveness to HCV, indicating that CD81 is essential for HCV infection and a defect in CD81 causes the nonpermissiveness to HCV in Huh7.5.1-5 cells. Huh7.5.1-8 cells had about 10-fold higher HCV replication activities, with cellular HCV RNA copy numbers of >10(9) copies/μg of cellular RNA and viral titers of >10(6) infectious units/ml of culture supernatant. Permissiveness of Huh7.5.1-8 cells to HCV infection was phenotypically very stable because there was no difference in permissiveness after more than 100 passages (1-year culture). This efficient cell culture system for HCV using Huh7.5.1-8 cells provides a powerful tool for studying the HCV life cycle and constructing antiviral strategies.
Full-text · Article · Nov 2014 · Japanese journal of infectious diseases
[Show abstract][Hide abstract]ABSTRACT: In recent decades, many sphingolipid enzymes, sphingolipid-metabolism regulators, and sphingolipid transfer proteins have been isolated and characterized. This review will provide an overview of the intracellular localization and topology of sphingolipid enzymes in mammalian cells to highlight the locations where respective sphingolipid species are produced. Interestingly, three sphingolipids that reside or are synthesized in cytosolic leaflets of membranes (ceramide, glucosylceramide and ceramide-1-phosphate) all have cytosolic lipid transfer proteins (LTPs). These LTPs consist of ceramide transfer protein (CERT), four-phosphate adaptor protein 2 (FAPP2) and ceramide-1-phosphate transfer protein (CPTP), respectively. These LTPs execute functions that affect both the location and metabolism of the lipids they bind. Molecular details describing the mechanisms of regulation of LTPs continue to emerge and reveal a number of critical processes, including competing phosphorylation and dephosphorylation reactions and binding interactions with regulatory proteins and lipids, that influence the transport, organelle distribution and metabolism of sphingolipids.
[Show abstract][Hide abstract]ABSTRACT: ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.
[Show abstract][Hide abstract]ABSTRACT: Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also
important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible
to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines
for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were
identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon
gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around
this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover,
a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the
non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines
and developing new tools in the quality control of Vero cells.
Full-text · Article · Sep 2014 · DNA research: an international journal for rapid publication of reports on genes and genomes
[Show abstract][Hide abstract]ABSTRACT: Mammalian cells store excess fatty acids in the form of triglycerides within lipid droplets. The intracellular bacterium Orientia tsutsugamush is the causative agent of severe human rickettiosis. We found that O. tsutsugamushi infection induces the formation of lipid droplets in mouse L-929 fibroblasts. In infected cells, a parallel increase in the number of lipid droplets and pathogens was observed. Interestingly, the pathogen-infection induced the accumulation of triglycerides even without external supply of fatty acids. These results suggest that O. tsutsugamushi alters lipid metabolism of host cells to induce lipid droplets.
No preview · Article · Sep 2014 · Microbes and Infection
[Show abstract][Hide abstract]ABSTRACT: Background
Progressive familial intrahepatic cholestasis type 1 (PFIC1), an inherited liver disease caused by mutations in ATP8B1, progresses to severe cholestasis with a sustained intractable itch. Currently, no effective therapy has been established for PFIC1. Decreased function of the bile salt export pump (BSEP) in hepatocytes is suggested to be responsible for the severe cholestasis observed in PFIC1. We found a previously unidentified pharmacological effect of 4-phenylbutyrate (4PB) that increases the expression and function of BSEP. Here, we tested 4PB therapy in three patients with PFIC1.
The therapeutic potency of 4PB in these patients was tested by oral administration of this drug with gradually increasing dosage (200, 350, and 500 mg/kg/day) for 6 months. Biochemical, histological, and clinical data were collected.
4PB therapy had no beneficial effect on the patients’ liver functions, as assessed by biochemical and histological analyses, despite an increase in hepatic BSEP expression. However, therapy with 4PB at a dosage of 350 or 500 mg/kg/day significantly relieved the intractable itch. Serum levels of potential pruritogens in cholestasis were much higher than the reference ranges during the 4PB therapy.
4PB therapy may be a new medication for patients with intractable cholestatic pruritus and may improve quality of life for patients and their families.
[Show abstract][Hide abstract]ABSTRACT: The nucleolar protein PICT1 regulates tumor suppressor p53 by tethering ribosomal protein L11 within the nucleolus to repress
the binding of L11 to the E3 ligase MDM2. PICT1 depletion results in the release of L11 to the nucleoplasm to inhibit MDM2,
leading to p53 activation. Here, we demonstrate that nucleolar stress induces proteasome-mediated degradation of PICT1 in
a ubiquitin-independent manner. Treatment of H1299 cells with nucleolar stress inducers, such as actinomycin D, 5-fluorouridine,
or doxorubicin, induced the degradation of PICT1 protein. The proteasome inhibitors MG132, lactacystin, and epoxomicin blocked
PICT1 degradation, whereas the inhibition of E1 ubiquitin-activating enzyme by a specific inhibitor and genetic inactivation
fail to repress PICT1 degradation. In addition, the 20 S proteasome was able to degrade purified PICT1 protein in vitro. We also found a PICT1 mutant showing nucleoplasmic localization did not undergo nucleolar stress-induced degradation, although
the same mutant underwent in vitro degradation by the 20 S proteasome, suggesting that nucleolar localization is indispensable for the stress-induced PICT1
degradation. These results suggest that PICT1 employs atypical proteasome-mediated degradation machinery to sense nucleolar
stress within the nucleolus.
Full-text · Article · Jun 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We here showed that the phosphorylation of CERT at serine-315 (S315), which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at S315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine rich motif; SRM) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the SRM and the phosphorylation of S315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.
Preview · Article · Feb 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract]ABSTRACT: Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs). A TALEN pair targeting the human CERT gene (alternative name COL4A3BP) encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase), and B4GalT5 (encoding the major lactosylceramide synthase), and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.
[Show abstract][Hide abstract]ABSTRACT: Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.
[Show abstract][Hide abstract]ABSTRACT: Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is critical for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires the yeast oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control the late stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.
Full-text · Article · Nov 2013 · Journal of Cell Science
[Show abstract][Hide abstract]ABSTRACT: Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the β isoform of class II PI3K (PI3K-C2β) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2β abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2β substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2β plays an indispensable role in the HCV replication cycle through the binding to HCV core protein.
Preview · Article · Sep 2013 · Biochemical and Biophysical Research Communications