Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood-borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze compartment-specific membrane fusion. Whereas most SNAREs are bona fide type II membrane proteins, Ykt6 lacks a proteinaceous membrane anchor but contains a prenylation consensus motif (CAAX box) and exists in an inactive cytosolic and an active membrane-bound form. We demonstrate that both forms are farnesylated at the carboxyl-terminal cysteine of the CCAIM sequence. Farnesylation is the prerequisite for subsequent palmitoylation of the upstream cysteine, which permits stable membrane association of Ykt6. The double-lipid modification and membrane association is crucial for intra-Golgi transport in vitro and cell homeostasis͞survival in vivo. The membrane recruitment and palmitoylation is controlled by the N-terminal domain of Ykt6, which interacts with the SNARE motif, keeping it in an inactive closed conformation. Together, these results suggest that conformational changes control the lipid modification and function of Ykt6. Considering the essential and central role of Ykt6 in the secretory pathway, this spatial and functional cycle might provide a mechanism to regulate the rate of intracellular membrane flow.T he dynamic and specific trafficking of proteins and lipids along the secretory and endocytic pathways relies on precisely choreographed membrane fusion events, in turn relying on the faithful pairing of cognate soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) between membranes (1, 2). In vitro membrane fusion assays employing purified SNAREs reconstituted into liposomes, as well as in vivo cell-cell fusion experiments using f lipped SNAREs [cognate-vesicle (v) and target-membrane (t) SNAREs expressed on the extracellular surface of two cell populations, respectively], provide compelling evidence that SNAREs are the driving force for fusion and, in addition, encode targeting specificity (3-7). Furthermore, as reported for intraGolgi transport, some SNAREs function also as inhibitory SNAREs to fine-tune specific fusion events (8). Thus, the intracellular distribution of cognate SNAREs outlines the fusion potential of distinct compartments and provides a road map for membrane trafficking (9, 10).The rate at which compartments containing cognate SNAREs fuse is at least in part determined by the conformational state of SNAREs. Most SNAREs contain regulatory domains in addition to their SNARE motifs, which form the four-helix bundle at the core of the SNARE complex (11). These regulatory domains control the assembly of the three-helix t-SNARE (one helix derived from a syntaxin heavy chain and two from t-SNARE light chains) and͞or the subsequent v-͞t-SNARE complex formation. Additional components can either bind or posttranslationally modify SNAREs, increasing or decreasing SNARE activity.A unique and therefore interesting SNARE is Ykt6, an essential protein that is highly conserved from yeast to man (12). Ykt6 is special in that it is lipid-anchored to membranes, lacking the usual hydroph...

In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.Hepatitis C virus (HCV) is recognized as a major cause of chronic liver disease, including chronic hepatitis, hepatic steatosis, cirrhosis, and hepatocellular carcinoma. It presently affects approximately 200 million people worldwide (26). HCV is an enveloped positive-strand RNA virus belonging to the Hepacivirus genus of the family Flaviviridae. Its genome of ϳ9.6 kb encodes a polyprotein precursor of ϳ3,000 residues, and the structural proteins (core, E1, and E2) reside in its N-terminal region.Little is known about the assembly of HCV and its virion structure, because efficient production of authentic HCV particles has only recently been achieved. Nucleocapsid assembly generally involves oligomerization of the capsid protein and encapsidation of genomic RNA. This process is thought to occur upon interaction of the core protein with viral RNA, and this core-RNA interaction may induce a change from RNA replication to packaging. As with related viruses, the mature HCV virion likely consists of a nucleocapsid and an outer envelope composed of a lipid membrane and envelope proteins. Expression of the structural proteins in mammalian cells has been observed to generate virus-like particles with ultrastructural properties similar to those of HCV virions (5, 29). Packaging of these HCV-like particles into intracellular vesicles as a result of budding from the endoplasmic reticulum (ER) has also been observed (8,34). However, HCV structural proteins are observed both in the ER and in the Golgi apparatus (45). Moreover, complex N-linked glycans have been detected on the surfaces of HCV particles isolated from patient sera, suggesting that the glycans transit through the Golgi apparatus (44). Interactions between the core and E1/E2 proteins are thought to determine viral morphology and are mediated thro...

Hepatitis C virus (HCV) is a positive-strand RNA virus, and classified within the Flaviridae family. Atg7-knockdown decreases the amount of HCV replicon RNA, when HCV JFH1 RNA and HCV subgenomic replicon are transfected into Huh7.5 cells. However, when infectious naive HCV particles are directly infected into Huh7.5.1 cells, it is still unclear whether Atg7-knockdown decreases the production of intracellular HCV-related proteins, HCV mRNA and infectious HCV particles. When Atg7 protein in HCV-infected Huh7.5.1 cells was knocked down by RNA-interference, the levels of intracellular HCV core, NS3, NS5A proteins, HCV mRNA and secreted albumin remained unchanged compared with those in the control HCV-infected cells. However, the level of infectious HCV particles released in the medium was decreased by the Atg7-knockdown. Similar results were obtained when Beclin 1 was knocked down by RNA-interference. The colocalization of endogenous LC3-puncta with HCV core, HS5A proteins and lipid droplets was also investigated. However, little endogenous LC3-puncta colocalized with HCV core, NS5A proteins or lipid droplets. These results suggested that autophagy contributed to the effective production of HCV particles, but little to the intracellular production of HCV-related proteins, HCV mRNA and the secretory pathway, in a naive HCV particles-infection system.