Metabolic channeling of lipids via the contact zones between different organelles.

Various lipid transfer proteins (LTPs) mediate the inter-organelle transport of lipids. By working at membrane contact zones between donor and acceptor organelles, LTPs achieve rapid and accurate inter-organelle transfer of lipids. This article will describe the emerging paradigm that the action of LTPs at organelle contact zones generates metabolic channeling events in lipid metabolism, mainly referring to how ceramide synthesized in the endoplasmic reticulum is preferentially metabolized to sphingomyelin in the distal Golgi region, how cholesterol and phospholipids receive specific metabolic reactions in mitochondria, and how the hijacking of host LTPs by intracellular pathogens may generate new channeling-like events. In addition, the article will discuss how the function of LTPs is regulated, exemplified by a few representative LTP systems, and will briefly touch on experiments that will be necessary to establish the paradigm that LTP-mediated inter-organelle transport of lipids is one of the mechanisms of compartmentalization-based metabolic channeling events.

Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3.

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

A high-resolution phase-contrast microscopy system for label-free imaging in living cells.

Cell biologists have long sought the ability to observe intracellular structures in living cells without labels. This study presents procedures to adjust a commercially available apodized phase-contrast (APC) microscopy system for better visualizing the dynamic behaviors of various subcellular organelles in living cells. By harnessing the versatility of this technique to capture sequential images, we could observe morphological changes in cellular geometry after virus infection in real time without probes or invasive staining. The tune-up APC microscopy system is a highly efficient platform for simultaneously observing the dynamic behaviors of diverse subcellular structures with exceptional resolution.

Establishment of measles virus receptor-expressing Vero cells lacking functional poliovirus receptors.

Global efforts are underway to eliminate measles and rubella, and active viral surveillance is the key to achieving this goal. In addition, the World Health Organization announced guidelines for handling materials potentially infectious for poliovirus (PV) to minimize the risk of PV reintroduction and to achieve PV eradication. To support global efforts, we established new PV-non-susceptible cell lines that are useful for the isolation of measles virus (MeV) and rubella virus (RuV) (Vero ΔPVR1/2 hSLAM+). In the cell lines, MeV and RuV replicated efficiently, with no concern regarding PV replication.

Organelle contacts: Sub-organelle zones to facilitate rapid and accurate inter-organelle trafficking of lipids.

When one person wants to communicate securely with another, he/she should contact the other person directly. This rule applies not only to human society, but also to the intracellular micro-society. In the past two decades, it has become increasingly clear that the sub-organelle regions called membrane contact sites (MCSs) are pivotal for inter-organelle transport of lipids in cells, as highlighted in the thematic review series "Interorganelle trafficking of lipids" held in Traffic in 2014-2015. In this commentary, we will describe how the currently prevailing model for lipid trafficking at MCSs was generated, and comment on three important issues that have not been explored: (a1) the principles guiding the generation of an asymmetrical inter-organelle flow of lipids in cells, (b2) the advantages in lipid trafficking at organelle contacts, and (c3) the dynamic network of inter-organelle lipid trafficking.

Nuclear Ceramide Is Associated with Ataxia Telangiectasia Mutated Activation in the Neocarzinostatin-Induced Apoptosis of Lymphoblastoid Cells.

Ceramide is a bioactive sphingolipid that mediates ionizing radiation- and chemotherapy-induced apoptosis. Neocarzinostatin (NCS) is a genotoxic anti-cancer drug that induces apoptosis in response to DNA double-strand breaks (DSBs) through ataxia telangiectasia mutated (ATM) activation. However, the involvement of ceramide in NCS-evoked nuclear events such as DSB-activated ATM has not been clarified. Here, we found that nuclear ceramide increased by NCS-mediated apoptosis through the enhanced assembly of ATM and the meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 (MRN) complex proteins in human lymphoblastoid L-39 cells. NCS induced an increase of ceramide production through activation of neutral sphingomyelinase (nSMase) and suppression of sphingomyelin synthase (SMS) upstream of DSB-mediated ATM activation. In ATM-deficient lymphoblastoid AT-59 cells compared with L-39 cells, NCS treatment showed a decrease of apoptosis even though ceramide increase and DSBs were observed. Expression of wild-type ATM, but not the kinase-dead mutant ATM, in AT-59 cells increased NCS-induced apoptosis despite similar ceramide accumulation. Interestingly, NCS increased ceramide content in the nucleus through nSMase activation and SMS suppression and promoted colocalization of ceramide with phosphorylated ATM and foci of MRN complex. Inhibition of ceramide generation by the overexpression of SMS suppressed NCS-induced apoptosis through the inhibition of ATM activation and assembly of the MRN complex. In addition, inhibition of ceramide increased by the nSMase inhibitor GW4869 prevented NCS-mediated activation of the ATM. Therefore, our findings suggest the involvement of the nuclear ceramide with ATM activation in NCS-mediated apoptosis. SIGNIFICANCE STATEMENT: This study demonstrates that regulation of ceramide with neutral sphingomyelinase and sphingomyelin synthase in the nucleus in double-strand break-mimetic agent neocarzinostatin (NCS)-induced apoptosis. This study also showed that ceramide increase in the nucleus plays a role in NCS-induced apoptosis through activation of the ataxia telangiectasia mutated/meiotic recombination 11/double-strand break repair/Nijmengen breakage syndrome 1 complex in human lymphoblastoid cells.

Intellectual-disability-associated mutations in the ceramide transport protein gene CERT1 lead to aberrant function and subcellular distribution.

The lipid molecule ceramide is transported from the endoplasmic reticulum to the Golgi apparatus for sphingomyelin production via the ceramide transport protein (CERT), encoded by CERT1. Hyperphosphorylation of CERT's serine-repeat motif (SRM) decreases its functionality. Some forms of inherited intellectual disability (ID) have been associated with a serine-to-leucine substitution in the SRM (S132L mutation) and a glycine-to-arginine substitution outside the SRM (G243R mutation) in CERT; however, it is unclear if mutations outside the SRM disrupt the control of CERT functionality. In the current investigation, we identified a new CERT1 variant (dupAA) in a patient with mild ID that resulted from a frameshift at the C-terminus of CERT1. However, familial analysis revealed that the dupAA variant was not associated with ID, allowing us to utilize it as a disease-matched negative control for CERT1 variants that are associated with ID. Biochemical analysis showed that G243R and S132L, but not dupAA, impair SRM hyperphosphorylation and render the CERT variants excessively active. Additionally, both S132L and G243R mutations but not dupAA caused the proteins to be distributed in a punctate subcellular manner. On the basis of these findings, we infer that the majority of ID-associated CERT variants may impair SRM phosphorylation-dependent repression, resulting in an increase in sphingomyelin production concurrent with CERT subcellular redistribution.

A hybrid strategy combining solution NMR spectroscopy and isothermal titration calorimetry to characterize protein-nanodisc interaction.

NMR is a powerful tool for characterizing intermolecular interactions at atomic resolution. However, the nature of the complex interactions of membrane-binding proteins makes it difficult to elucidate the interaction mechanisms. Here, we demonstrated that structural and thermodynamic analyses using solution NMR spectroscopy and isothermal titration calorimetry (ITC) can clearly detect a specific interaction between the pleckstrin homology (PH) domain of ceramide transport protein (CERT) and phosphatidylinositol 4-monophosphate (PI4P) embedded in the lipid nanodisc, and distinguish the specific interaction from nonspecific interactions with the bulk surface of the lipid nanodisc. This NMR-ITC hybrid strategy provides detailed characterization of protein-lipid membrane interactions.

Natural Ligand-Mimetic and Nonmimetic Inhibitors of the Ceramide Transport Protein CERT.

Lipid transfer proteins (LTPs) are recognized as key players in the inter-organelle trafficking of lipids and are rapidly gaining attention as a novel molecular target for medicinal products. In mammalian cells, ceramide is newly synthesized in the endoplasmic reticulum (ER) and converted to sphingomyelin in the trans-Golgi regions. The ceramide transport protein CERT, a typical LTP, mediates the ER-to-Golgi transport of ceramide at an ER-distal Golgi membrane contact zone. About 20 years ago, a potent inhibitor of CERT, named (1R,3S)-HPA-12, was found by coincidence among ceramide analogs. Since then, various ceramide-resembling compounds have been found to act as CERT inhibitors. Nevertheless, the inevitable issue remains that natural ligand-mimetic compounds might directly bind both to the desired target and to various undesired targets that share the same natural ligand. To resolve this issue, a ceramide-unrelated compound named E16A, or (1S,2R)-HPCB-5, that potently inhibits the function of CERT has recently been developed, employing a series of in silico docking simulations, efficient chemical synthesis, quantitative affinity analysis, protein-ligand co-crystallography, and various in vivo assays. (1R,3S)-HPA-12 and E16A together provide a robust tool to discriminate on-target effects on CERT from off-target effects. This short review article will describe the history of the development of (1R,3S)-HPA-12 and E16A, summarize other CERT inhibitors, and discuss their possible applications.

Hyperosmotic Stress Induces Phosphorylation of CERT and Enhances Its Tethering throughout the Endoplasmic Reticulum.

The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER-Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER-Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM.

Chlamydial Infection-Dependent Synthesis of Sphingomyelin as a Novel Anti-Chlamydial Target of Ceramide Mimetic Compounds.

The obligate intracellular bacterium Chlamydia trachomatis is the major causative agent of bacterial sexually transmitted diseases worldwide. In infected cells, the ceramide transport protein (CERT) is recruited to inclusions, where C. trachomatis replicates using host-synthesized ceramide. The ceramide is converted to sphingomyelin (SM) by a chlamydial infection-dependent SM synthesis (cidSM-synthesis) pathway, which occurs even in the absence of the SM synthases (SMS)-1 and -2 of host cells. The ceramide mimetic compound (1R,3S)-HPA-12 and the nonmimetic compound E16A, both of which are potent inhibitors of CERT, repressed the proliferation of C. trachomatis in HeLa cells. Unexpectedly, (1R,3R)-HPA-12, a ceramide mimetic compound that lacks CERT inhibitory activity, also exhibited potent anti-chlamydial activity. Using endogenous SMS-knockout mutant HeLa cells, we revealed that (1R,3R)-HPA-12 mildly inhibited cidSM-synthesis. In addition, LC-MS analysis revealed that (1R,3R)-HPA-12 is converted to a phosphocholine-conjugated metabolite in an infection-dependent manner. Imaging analysis with a fluorescent analog of ceramide suggested that cidSM-synthesis occurs in the bacterial bodies and/or inclusions. Collectively, these results suggested that (1R,3R)-HPA-12 exerts its anti-chlamydia activity not only as an inhibitor of cidSM-synthesis, but also via putative toxic effects of its phosphocholine adduct, which is most likely produced by the cidSM-synthesis route.

Involvement of a Cluster of Basic Amino Acids in Phosphorylation-Dependent Functional Repression of the Ceramide Transport Protein CERT.

Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.

Blood group P1 antigen-bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors.

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galß1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galß1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.

Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories.

Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses.IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

Structural basis of antiviral activity of caffeic acid against severe fever with thrombocytopenia syndrome virus.

Caffeic acid (CA), a coffee-related natural compound, has various beneficial biological effects, including antiviral effects. Our former studies demonstrated that the CA dose-dependently inhibited the in vitro infection with Dabie bandavirus, which was previously named as severe fever with thrombocytopenia syndrome virus (SFTSV), mainly at the step of virus attachment. Therefore, we studied the structural basis of CA for conferring anti-SFTSV activity to clarify the mechanism of action of CA against SFTSV. In this study, the anti-SFTSV activity of nine CA analogs were examined. The treatment of SFTSV with the 3,4-dihydroxyhydrocinnamic acid (DHCA) as well as CA inhibited the SFTSV infection in a dose-dependent manner, whereas other CA analogs did not. Both CA and DHCA only possessed the o-dihydroxybenzene backbone. When SFTSV was treated with catechol (o-dihydroxybenzene), SFTSV infection was also dose-dependently inhibited. Additionally, four compounds having the o-dihydroxybenzene backbone; CA phenethyl ester, methyl CA, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxybenzoic acid, dose-dependently inhibited the viral infection, although these compounds were more toxic or less effective than CA. In conclusion, the o-dihydroxybenzene backbone in CA and its analogs was a critical structure for the anti-SFTSV activity. Based on these findings, modifications of the o-dihydroxybenzene backbone with various other residues might improve the antiviral effect and cytotoxicity for SFTSV.

Identification of SYS1 as a Host Factor Required for Shiga Toxin-Mediated Cytotoxicity in Vero Cells.

Shiga toxin (STx) or Vero toxin is a virulence factor produced by enterohemorrhagic Escherichia coli. The toxin binds to the glycosphingolipid globotriaosylceramide (Gb3) for its entry, and causes cell death by inhibiting ribosome function. Previously, we performed a loss-of-function screen in HeLa cells using a human CRISPR knockout (KO) library and identified various host genes required for STx-induced cell death. To determine whether this library targeted to the human genome is applicable to non-human primate cells and to identify previously unrecognized factors crucial for STx-induced cell death, we herein performed a similar screen in the African green monkey kidney-derived Vero C1008 subline. Many genes relevant to metabolic enzymes and membrane trafficking were enriched, although the number of enriched genes was less than that obtained in the screening for HeLa cells. Of note, several genes that had not been enriched in the previous screening were enriched: one of these genes was SYS1, which encodes a multi-spanning membrane protein in the Golgi apparatus. In SYS1 KO Vero cells, expression of Gb3 and sphingomyelin was decreased, while that of glucosylceramide and lactosylceramide was increased. In addition, loss of SYS1 inhibited the biosynthesis of protein glycans, deformed the Golgi apparatus, and perturbed the localization of trans-Golgi network protein (TGN) 46. These results indicate that the human CRISPR KO library is applicable to Vero cell lines, and SYS1 has a widespread effect on glycan biosynthesis via regulation of intra-Golgi and endosome-TGN retrograde transports.

Phosphoinositide binding by the PH domain in ceramide transfer protein (CERT) is inhibited by hyperphosphorylation of an adjacent serine-repeat motif.

Sphingolipids such as ceramide are important constituents of cell membranes. The ceramide transfer protein (CERT) moves ceramide from the endoplasmic reticulum to the Golgi apparatus in a nonvesicular manner. Hyperphosphorylation of the serine-repeat motif (SRM) adjacent to the pleckstrin homology (PH) domain of CERT down-regulates the inter-organelle ceramide transport function of CERT. However, the mechanistic details of this down-regulation remain elusive. Using solution NMR and binding assays, we herein show that a hyperphosphorylation-mimetic CERT variant in which 10 serine/threonine residues of SRM had been replaced with glutamate residues (the 10E variant) displays an intramolecular interaction between SRM and positively charged regions of the PH domain, which are involved in the binding of this domain to phosphatidylinositol 4-monophosphate (PI4P). Of note, the binding of the PH domain to PI4P-embedded membranes was attenuated by the SRM 10E substitutions in cell-free assays. Moreover, the 10E substitutions reduced the Golgi-targeting activity of the PH-SRM construct in living cells. These results indicate that hyperphosphorylated SRM directly interacts with the surface of the PH domain in an intramolecular manner, thereby decreasing the PI4P-binding activity of the PH domain. In light of these findings, we propose that the hyperphosphorylation of SRM may trigger the dissociation of CERT from the Golgi apparatus, resulting in a functionally less active conformation of CERT.

Pleckstrin homology domain of p210 BCR-ABL interacts with cardiolipin to regulate its mitochondrial translocation and subsequent mitophagy.

Chronic myeloid leukemia (CML) is caused by the chimeric protein p210 BCR-ABL encoded by a gene on the Philadelphia chromosome. Although the kinase domain of p210 BCR-ABL is an active driver of CML, the pathological role of its pleckstrin homology (PH) domain remains unclear. Here, we carried out phospholipid vesicle-binding assays to show that cardiolipin (CL), a characteristic mitochondrial phospholipid, is a unique ligand of the PH domain. Arg726, a basic amino acid in the ligand-binding region, was crucial for ligand recognition. A subset of wild-type p210 BCR-ABL that was transiently expressed in HEK293 cells was dramatically translocated from the cytosol to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, which induces mitochondrial depolarization and subsequent externalization of CL to the organelle's outer membrane, whereas an R726A mutant of the protein was not translocated. Furthermore, only wild-type p210 BCR-ABL, but not the R726A mutant, suppressed CCCP-induced mitophagy and subsequently enhanced reactive oxygen species production. Thus, p210 BCR-ABL can change its intracellular localization via interactions between the PH domain and CL to cope with mitochondrial damage. This suggests that p210 BCR-ABL could have beneficial effects for cancer proliferation, providing new insight into the PH domain's contribution to CML pathogenesis.

Both Sphingomyelin and Cholesterol in the Host Cell Membrane Are Essential for Rubella Virus Entry.

Rubella virus (RuV) causes a systemic infection, and transplacental fetal infection causes congenital rubella syndrome. In this study, we showed that treatment of cells with sphingomyelinase inhibited RuV infection. Assays using inhibitors of serine palmitoyl transferase and ceramide transport protein demonstrated the contribution of sphingomyelin (SM) to RuV infection. Compelling evidence for direct binding of RuV to lipid membranes at neutral pH was obtained using liposome coflotation assays. The absence of either SM or cholesterol (Chol) abrogated the RuV-liposome interaction. SM and Chol (SM/Chol) were also critical for RuV binding to erythrocytes and lymphoid cells. Removal of Ca2+ from the assay buffer or mutation of RuV envelope E1 protein Ca2+-binding sites abrogated RuV binding to liposomes, erythrocytes, and lymphoid cells. However, RuV bound to various nonlymphoid adherent cell lines independently of extracellular Ca2+ or SM/Chol. Even in these adherent cell lines, both the E1 protein Ca2+-binding sites and cellular SM/Chol were essential for the early stage of RuV infection, possibly affecting envelope-membrane fusion in acidic compartments. Myelin oligodendrocyte glycoprotein (MOG) has recently been identified as a cellular receptor for RuV. However, RuV bound to MOG-negative cells in a Ca2+-independent manner. Collectively, our data demonstrate that RuV has two distinct binding mechanisms: one is Ca2+ dependent and the other is Ca2+ independent. Ca2+-dependent binding observed in lymphoid cells occurs by the direct interaction between E1 protein fusion loops and SM/Chol-enriched membranes. Clarification of the mechanism of Ca2+-independent RuV binding is an important next step in understanding the pathology of RuV infection.IMPORTANCE Rubella has a significant impact on public health as infection during early pregnancy can result in babies being born with congenital rubella syndrome. Even though effective rubella vaccines are available, rubella outbreaks still occur in many countries. We studied the entry mechanism of rubella virus (RuV) and found that RuV binds directly to the host plasma membrane in the presence of Ca2+ at neutral pH. This Ca2+-dependent binding is specifically directed to membranes enriched in sphingomyelin and cholesterol and is critical for RuV infection. Importantly, RuV also binds to many cell lines in a Ca2+-independent manner. An unidentified RuV receptor(s) is involved in this Ca2+-independent binding. We believe that the data presented here may aid the development of the first anti-RuV drug.

Monoclonal Antibodies against Occludin Completely Prevented Hepatitis C Virus Infection in a Mouse Model.

Hepatitis C virus (HCV) entry into host cells is a multistep process requiring various host factors, including the tight junction protein occludin (OCLN), which has been shown to be essential for HCV infection in in vitro cell culture systems. However, it remains unclear whether OCLN is an effective and safe target for HCV therapy, owing to the lack of binders that can recognize the intact extracellular loop domains of OCLN and prevent HCV infection. In this study, we successfully generated four rat anti-OCLN monoclonal antibodies (MAbs) by the genetic immunization method and unique cell differential screening. These four MAbs bound to human OCLN with a very high affinity (antibody dissociation constant of <1 nM). One MAb recognized the second loop of human and mouse OCLN, whereas the three other MAbs recognized the first loop of human OCLN. All MAbs inhibited HCV infection in Huh7.5.1-8 cells in a dose-dependent manner without apparent cytotoxicity. Additionally, the anti-OCLN MAbs prevented both cell-free HCV infection and cell-to-cell HCV transmission. Kinetic studies with anti-OCLN and anti-claudin-1 (CLDN1) MAbs demonstrated that OCLN interacts with HCV after CLDN1 in the internalization step. Two selected MAbs completely inhibited HCV infection in human liver chimeric mice without apparent adverse effects. Therefore, OCLN would be an appropriate host target for anti-HCV entry inhibitors, and anti-OCLN MAbs may be promising candidates for novel anti-HCV agents, particularly in combination with direct-acting HCV antiviral agents.IMPORTANCE HCV entry into host cells is thought to be a very complex process involving various host entry factors, such as the tight junction proteins claudin-1 and OCLN. In this study, we developed novel functional MAbs that recognize intact extracellular domains of OCLN, which is essential for HCV entry into host cells. The established MAbs against OCLN, which had very high affinity and selectivity for intact OCLN, strongly inhibited HCV infection both in vitro and in vivo Using these anti-OCLN MAbs, we found that OCLN is necessary for the later stages of HCV entry. These anti-OCLN MAbs are likely to be very useful for understanding the OCLN-mediated HCV entry mechanism and might be promising candidates for novel HCV entry inhibitors.