C-terminal Glycosylphosphatidylinositol Research Articles

GPI-anchored Gas1 protein regulates cytosolic proteostasis in budding yeast.

The decline in protein homeostasis (proteostasis) is a hallmark of cellular aging and aging-related diseases. Maintaining a balanced proteostasis requires a complex network of molecular machineries that govern protein synthesis, folding, localization, and degradation. Under proteotoxic stress, misfolded proteins that accumulate in cytosol can be imported into mitochondria for degradation through the "mitochondrial as guardian in cytosol" (MAGIC) pathway. Here, we report an unexpected role of Gas1, a cell wall-bound glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase in the budding yeast, in differentially regulating MAGIC and ubiquitin-proteasome system (UPS). Deletion of GAS1 inhibits MAGIC but elevates protein ubiquitination and UPS-mediated protein degradation. Interestingly, we found that the Gas1 protein exhibits mitochondrial localization attributed to its C-terminal GPI anchor signal. But this mitochondria-associated GPI anchor signal is not required for mitochondrial import and degradation of misfolded proteins through MAGIC. By contrast, catalytic inactivation of Gas1 via the gas1-E161Q mutation inhibits MAGIC but not its mitochondrial localization. These data suggest that the glucanosyltransferase activity of Gas1 is important for regulating cytosolic proteostasis.

Glycosylphosphatidylinositol-anchoring is required for the proper transport and extensive glycosylation of a classical arabinogalactan protein precursor in tobacco BY-2 cells.

Many precursors of plant arabinogalactan proteins (AGPs) contain a C-terminal glycosylphosphatidylinositol (GPI)-anchoring signal. Using NtAGP1, a classical tobacco AGP, as a model, and green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags, we analyzed the localization and modification of AGP and its mutant without GPI-anchoring signal (AGPΔC) in tobacco BY-2 cells. The NtAGP1 fusion proteins migrated as large smear on SDS-polyacrylamide gel, and these proteins also localized preferentially to the plasma membrane. In contrast, fusions of AGPΔC with GFP and SPO yielded several forms: The largest were secreted, whereas others were recovered in the endomembrane organelles, including vacuoles. Comparison of the glycan structures of the microsomal SPO-AGP and the secreted SPO-AGPΔC using antibodies against the glycan epitopes of AGP indicated that the glycan structures of these proteins are different. These observations indicate that GPI-anchoring is required for the proper transport and glycosylation of the AGP precursor.

Structural analysis of Plasmodium falciparum ookinete surface antigen Pfs28 relevant for malaria vaccine design

Pfs28 is a Plasmodium falciparum malaria transmission-blocking vaccine candidate that is anchored to the parasite surface through a C-terminal glycosylphosphatidylinositol (GPI) moiety, and plays a role in parasite survival in the mosquito midgut. Pfs28 contains epidermal growth factor (EGF)-like domains and is part of a family of sexual stage malaria proteins that includes the related vaccine antigen Pfs25. The lack of structural definition of Pfs28 and the immune response to this candidate has limited further malaria vaccine development for this antigen. Here, we present the crystal structure of Pfs28, examine its conservation with P. vivax Pvs28, and evaluate the cross-reactivity of Pfs28 to antibodies that recognize Pfs25. Pfs28 is comprised of four EGF-like domains stabilized by ten disulfide bridges with an overall architecture that highly resembles Pfs25. Despite the high sequence and structural similarity between these antigens, no cross reactivity of Pfs28 to anti-Pfs25 monoclonal antibodies could be demonstrated.

Identification and functional characterization of complement regulatory protein CD59 in golden pompano (Trachinotus ovatus)

CD59, one of the essential inhibitors of the complement membrane attack complex (MAC), plays a crucial role in regulation of complement activation. In this study, we cloned and identified the CD59 gene (named ToCD59) of golden pompano (Trachinotus ovatus). The ORF sequence of ToCD59 is 357 bp long encoding 118 amino acids with a molecular weight of 13.09 kDa. Prediction of protein domains showed that ToCD59 contained an Lu domain and a C-terminal glycosylphosphatidylinositol (GPI) partial anchor. Homology comparisons indicated that ToCD59 shared the high sequence similarity with other fish CD59. RT-qPCR analysis showed that ToCD59 was expressed in all tested healthy tissues of golden pompano, with the highest level of expression in the brain. After stimulation with bacteria, ToCD59 expression levels were significantly up-regulated in head kidney, liver, gill and brain, but down-regulated in spleen. Subcellular localization results showed that ToCD59 localized to the cytoplasm of A549 cells. The hemolytic activity analysis showed that rToCD59 might have complement inhibitory activity through the alternative complement pathway. In addition, antibacterial test showed that rToCD59 had antibacterial ability against S. agalactiae and V. alginolyticus in vitro. These results suggest that ToCD59 might play an important role in the immune response against pathogens, which would provide basic information for elucidating the functional evolutionary history of complement system in teleost.

Repulsive guidance molecule acts in axon branching in Caenorhabditis elegans

Repulsive guidance molecules (RGMs) are evolutionarily conserved proteins implicated in repulsive axon guidance. Here we report the function of the Caenorhabditis elegans ortholog DRAG-1 in axon branching. The axons of hermaphrodite-specific neurons (HSNs) extend dorsal branches at the region abutting the vulval muscles. The drag-1 mutants exhibited defects in HSN axon branching in addition to a small body size phenotype. DRAG-1 expression in the hypodermal cells was required for the branching of the axons. Although DRAG-1 is normally expressed in the ventral hypodermis excepting the vulval region, its ectopic expression in vulval precursor cells was sufficient to induce the branching. The C-terminal glycosylphosphatidylinositol anchor of DRAG-1 was important for its function, suggesting that DRAG-1 should be anchored to the cell surface. Genetic analyses suggested that the membrane receptor UNC-40 acts in the same pathway with DRAG-1 in HSN branching. We propose that DRAG-1 expressed in the ventral hypodermis signals via the UNC-40 receptor expressed in HSNs to elicit branching activity of HSN axons.

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit.

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Non‐enzymatic function of prostasin and sodium balance

Prostasin is a serine protease with a simple structure: a serine protease domain attached to the cell surface via a C-terminal glycosylphosphatidylinositol (GPI) anchor. Several physiological processes require membrane-attached prostasin, but, surprisingly, without its protease activity. In the current issue of Acta Physiologica, Essigke and colleagues 1 suggest that this is also the case for prostasin's activation of the epithelial Na+ channel ENaC in the kidney.

Diversity of GPI-anchored fungal adhesins.

Selective adhesion of fungal cells to one another and to foreign surfaces is fundamental for the development of multicellular growth forms and the successful colonization of substrates and host organisms. Accordingly, fungi possess diverse cell wall-associated adhesins, mostly large glycoproteins, which present N-terminal adhesion domains at the cell surface for ligand recognition and binding. In order to function as robust adhesins, these glycoproteins must be covalently linkedto the cell wall via C-terminal glycosylphosphatidylinositol (GPI) anchors by transglycosylation. In this review, we summarize the current knowledge on the structural and functional diversity of so far characterized protein families of adhesion domains and set it into a broad context by an in-depth bioinformatics analysis using sequence similarity networks. In addition, we discuss possible mechanisms for the membrane-to-cell wall transfer of fungal adhesins by membrane-anchored Dfg5 transglycosidases.

Genome-wide identification of fasciclin-like arabinogalactan proteins in jute and their expression pattern during fiber formation.

Fasciclin-like arabinogalactan proteins (FLAs), a class of arabinogalactan proteins (AGPs) are involved in plant growth and development via cell communication and adhesion. FLAs were also associated with fiber and wood formation in plants but no information is available about the roles of FLA proteins during fibre development of jute. Here, we performed molecular characterization, evolutionary relationship and expression profiling of FLAs proteins in jute (Corchorus olitorius). In total, nineteen CoFLA genes have been identified in jute genome, which were divided into four classes like FLAs of other species based on protein structure and similarity. All CoFLAs have N-terminal signal peptide and one or two FAS domain while two FLAs lack well defined AGP region and eight FLAs were devoid of C-terminal glycosylphosphatidylinositol (GPI) anchor. Expression analysis of different regions of jute stem suggested their involvement in different fiber development stages. Four genes CoFLA 11, 12, 20, and 23 were highly or predominately expressed in fiber containing bark tissues while the expression levels of six CoFLA genes 02, 03, 04, 06, 14 and 19 were comparatively higher in stick. Higher transcripts levels of CoFLA 12 and 20 in the middle bark tissues suggest their involvement in fiber elongation. In contrast, the CoFLA 11 and 23 were more expressed in bottom bark tissues suggesting their potential involvement in secondary cell wall synthesis. Our study can serve as solid foundation for further functional exploration of FLAs and in future breeding program of jute aiming fiber improvement.

Identification of yeast-derived emulsification proteins through analyses of proteins distributed into the emulsified phase

Emulsifiers are widely used in food manufacturing, and cell wall mannoproteins from Saccharomyces cerevisiae, a representative food yeast, have been proposed as potential new emulsifiers. However, their use has not become widespread. One reason is that the protein responsible for emulsification has not been identified so no efficient preparation method has been established. In this study, the emulsification substances were identified. Many mannoproteins are fixed to the cell wall via a glycosylphosphatidylinositol (GPI) anchor. Because emulsifying activity was found in the culture supernatant (CS) in gup1Δ with a mutation of GPI anchor synthesis, a protein distributed from the CS into the emulsified phase by emulsification treatment was analyzed. Using mass spectrometry, it was identified to be Gas1, a mannoprotein localized to the cell wall via a GPI anchor. Further, recombinant Gas1 without a C-terminal GPI anchor binding signal was purified, and it showed activity similar to bovine serum albumin, a well-known protein emulsifier. Gas1 also retained its activity under a wide range of pH and high salt conditions. Saccharomyces cerevisiae has Gas3 and Gas5, proteins homologous to Gas1, and recombinant Gas3 and Gas5 without the C-terminal signal were also purified and found to have emulsifying activity similar to that of recombinant Gas1. These results strongly suggest that Gas proteins take part in the emulsifying activity of yeast mannoproteins. It was also demonstrated that emulsification proteins could be identified by analyzing proteins distributed in an emulsified phase, so this method will be effective in finding new emulsification proteins.

HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain

HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.

BST2/Tetherin Overexpression Modulates Morbillivirus Glycoprotein Production to Inhibit Cell-Cell Fusion.

The measles virus (MeV), a member of the genus Morbillivirus, is an established pathogen of humans. A key feature of morbilliviruses is their ability to spread by virus–cell and cell–cell fusion. The latter process, which leads to syncytia formation in vitro and in vivo, is driven by the viral fusion (F) and haemagglutinin (H) glycoproteins. In this study, we demonstrate that MeV glycoproteins are sensitive to inhibition by bone marrow stromal antigen 2 (BST2/Tetherin/CD317) proteins. BST2 overexpression causes a large reduction in MeV syncytia expansion. Using quantitative cell–cell fusion assays, immunolabeling, and biochemistry we further demonstrate that ectopically expressed BST2 directly inhibits MeV cell–cell fusion. This restriction is mediated by the targeting of the MeV H glycoprotein, but not other MeV proteins. Using truncation mutants, we further establish that the C-terminal glycosyl-phosphatidylinositol (GPI) anchor of BST2 is required for the restriction of MeV replication in vitro and cell–cell fusion. By extending our study to the ruminant morbillivirus peste des petits ruminants virus (PPRV) and its natural host, sheep, we also confirm this is a broad and cross-species specific phenotype.

Metabolic Labeling and Structural Analysis of Glycosylphosphatidylinositols from Parasitic Protozoa.

Glycosylphosphatidylinositol (GPI) is a complex glycolipid structure that acts as a membrane anchor for many cell-surface proteins of eukaryotes. GPI-anchored proteins are particularly abundant in protozoa and represent the major carbohydrate modification of many cell-surface parasite proteins. A minimal GPI-anchor precursor consists of core glycan (ethanolamine-PO4-Manα1-2Manα1-6Manα1-4GlcNH2) linked to the 6-position of the D-myo-inositol ring of phosphatidylinositol. Although the GPI core glycan is conserved in all organisms, many differences in additional modifications to GPI structures and biosynthetic pathways have been reported. The preassembled GPI-anchor precursor is post-translationally transferred to a variety of membrane proteins in the lumen of the endoplasmic reticulum in a transamidase-like reaction during which a C-terminal GPI attachment signal is released. Increasing evidence shows that a significant proportion of the synthesized GPIs are not used for protein anchoring, particularly in protozoa in which a large amount of free GPIs are being displayed at the cell surface. The characteristics of GPI biosynthesis are currently being explored for the development of parasite-specific inhibitors. Especially this pathway, at least for Trypanosoma brucei, has been validated as a drug target. Furthermore, thanks to an increase of new innovative strategies to produce pure synthetic carbohydrates, a novel era in the use of GPIs in diagnostic, anti-GPI antibody production, as well as parasitic protozoa GPI-based vaccine approach is developing fast.

PrP Knockout Cells Expressing Transmembrane PrP Resist Prion Infection.

Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres We show that only GPI-anchored PrPC was able to convert to PrPres and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPC to rafts are crucial to the ability of PrPC to propagate as a prion.

Kunitz 3-Selective but Not Kunitz 2-Selective TFPI Inhibitors Cause Release of Tfpiα from Endothelial Cells: Insights into the Release Mechanism

BACKGROUNDTissue factor pathway inhibitor (TFPI) is an anticoagulant protein that regulates early blood coagulation events and contains 3 Kunitz-type serine protease domains (K1-3). TFPI inhibitors are being developed to treat hemophilia. However, an inhibitory aptamer to the K3 domain of TFPI has been found to produce an increase in plasma TFPI with associated bleeding. Therefore, understanding the mechanisms causing release of TFPI from endothelium is important for further development of these drugs. Two major isoforms of TFPI, TFPIα and TFPIβ, are generated by human endothelial cells. Both isoforms contain K1 and K2. TFPIβ attaches to the cell surface via a C-terminal glycosylphosphatidyl inositol (GPI) anchor. TFPIα is a soluble protein with an additional Kunitz domain (K3) and a basic C-terminal tail present in human plasma. It has been found that plasma TFPIα promptly increases 2- to 4-fold following heparin infusion. Treatment of cultured human endothelial cells with heparin also promptly increases TFPIα 2- to 4-fold in culture media, suggesting that the basic C-terminal tail of TFPIα interacts with cell surface glycosaminoglycans (GAGs) and that this association is disrupted by heparin, releasing TFPIα into plasma or media. However, cell surface TFPI can be almost entirely removed by treatment with phosphatidylinositol phospholipase C, an enzyme that removes GPI-anchored proteins from the cell surface, indicating that only the GPI-anchored TFPIβ is present at the surface. This suggests that heparin may cause release of TFPIα from an intracellular pool. Thus, the mechanism of heparin-induced TFPIα release from endothelium remains unresolved. METHODSWild type (WT) and aerolysin-resistant (AR) Ea.hy926 cells, which lack GPI-anchored proteins, including TFPIβ, but produce normal amounts of TFPIα, were used to examine how heparin and anti-TFPI antibodies induce release of TFPI. For this, WT or AR cells were incubated for 15 minutes at room temperature or 4°C, with either heparin, 0.3 M NaCl, anti-K2 or anti-K3 antibody. Surface TFPI was detected with an anti-K2 antibody using flow cytometry. TFPIα released into culture media was measured with a TFPIα-specific ELISA. Brefeldin A, an inhibitor of protein transport from endoplasmic reticulum to Golgi, was used to block TFPIα secretion. Real time PCR was used to measure changes in TFPI mRNA. RESULTSWT cells express surface TFPI that can be detected with an anti-K2 antibody using flow cytometry and which is not altered by exposure of cells to heparin or anti-K3 antibody. AR cells do not have TFPI on their surface that is detectable by flow cytometry. Incubation of WT or AR cells with heparin, 0.3 M NaCl, or anti-K3 antibody produced an approximately 4-fold increase of TFPIα released into culture media. This was not observed when cells were incubated with anti-K2 antibody. To determine if heparin induces release of TFPIα from inside the cell, they were incubated for 15 minutes with heparin 4 times, in the presence or absence of Brefeldin A. During the fourth 15 minute treatment the amount of TFPIα secreted into the media was equal in heparin-treated or control cells. The cells were then allowed to incubate for an additional hour in the presence or absence of heparin. During this time, heparin-treated cells secreted TFPIα at a 2-fold faster rate than cells in buffer only (p=0.036). This difference was not observed in cells treated with Brefeldin A, which reduced TFPIα secretion by almost 5-fold (p=0.005). TFPIα transcript levels were not different between the heparin- and non-heparin-treated cells. CONCLUSIONTFPIα is released from Ea.hy926 cells by heparin and an anti-K3 antibody, both of which bind to the C-terminal region of TFPIα, while an anti-K2 antibody has been shown not to cause release of TFPIα from the cells. Indirect evidence suggests that there is a pool of TFPIα associated with cell surface GAGs and that this pool is released by agents that disrupt this interaction. However, the presence of TFPIα on the cell surface cannot be directly demonstrated using flow cytometry. Cells stripped of heparin-releasable TFPIα continue to secrete it into the media at a faster rate in the presence of heparin. Heparin does not alter transcription of TFPIα. Instead, it appears to enhance its secretion, perhaps by diverting it from storage in an intracellular pool. DisclosuresAugustsson:Novo Nordisk: Employment. Heibroch Petersen:Novo Nordisk: Employment. Hilden:Novo Nordisk: Employment. Mast:Novo Nordisk: Research Funding; Siemens: Honoraria.

Early Vertebrate Evolution of the Host Restriction Factor Tetherin.

Tetherin is an interferon-inducible restriction factor targeting a broad range of enveloped viruses. Its antiviral activity depends on an unusual topology comprising an N-terminal transmembrane domain (TMD) followed by an extracellular coiled-coil region and a C-terminal glycosylphosphatidylinositol (GPI) anchor. One of the two membrane anchors is inserted into assembling virions, while the other remains in the plasma membrane of the infected cell. Thus, tetherin entraps budding viruses by physically bridging viral and cellular membranes. Although tetherin restricts the release of a large variety of diverse human and animal viruses, only mammalian orthologs have been described to date. Here, we examined the evolutionary origin of this protein and demonstrate that tetherin orthologs are also found in fish, reptiles, and birds. Notably, alligator tetherin efficiently blocks the release of retroviral particles. Thus, tetherin emerged early during vertebrate evolution and acquired its antiviral activity before the mammal/reptile divergence. Although there is only limited sequence homology, all orthologs share the typical topology. Two unrelated proteins of the slime mold Dictyostelium discoideum also adopt a tetherin-like configuration with an N-terminal TMD and a C-terminal GPI anchor. However, these proteins showed no evidence for convergent evolution and failed to inhibit virion release. In summary, our findings demonstrate that tetherin emerged at least 450 million years ago and is more widespread than previously anticipated. The early evolution of antiviral activity together with the high topology conservation but low sequence homology suggests that restriction of virus release is the primary function of tetherin. The continuous arms race with viruses has driven the evolution of a variety of cell-intrinsic immunity factors that inhibit different steps of the viral replication cycle. One of these restriction factors, tetherin, inhibits the release of newly formed progeny virions from infected cells. Although tetherin targets a broad range of enveloped viruses, including retro-, filo-, herpes-, and arenaviruses, the evolutionary origin of this restriction factor and its antiviral activity remained obscure. Here, we examined diverse vertebrate genomes for genes encoding cellular proteins that share with tetherin the highly unusual combination of an N-terminal transmembrane domain and a C-terminal glycosylphosphatidylinositol anchor. We show that tetherin orthologs are found in fish, reptiles, and birds and demonstrate that alligator tetherin efficiently inhibits the release of retroviral particles. Our findings identify tetherin as an evolutionarily ancient restriction factor and provide new important insights into the continuous arms race between viruses and their hosts.

The Interferon-Inducible Protein Tetherin Inhibits Hepatitis B Virus Virion Secretion.

Interferon alpha (IFN-α) is an approved medication for chronic hepatitis B therapy. Besides acting as an immunomodulator, IFN-α elicits a pleiotropic antiviral state in hepatitis B virus (HBV)-infected hepatocytes, but whether or not IFN-α impedes the late steps of the HBV life cycle, such as HBV secretion, remains elusive. Here we report that IFN-α treatment of HepAD38 cells with established HBV replication selectively reduced HBV virion release without altering intracellular viral replication or the secretion of HBV subviral particles and nonenveloped capsids. In search of the interferon-stimulated gene(s) that is responsible for the reduction of HBV virion release, we found that tetherin, a broad-spectrum antiviral transmembrane protein that inhibits the egress of a variety of enveloped viruses, was highly induced by IFN-α in HepAD38 cells and in primary human hepatocytes. We further demonstrated that the expression of full-length tetherin, but not the C-terminal glycosylphosphatidylinositol (GPI) anchor-truncated form, inhibited HBV virion egress from HepAD38 cells. In addition, GPI anchor-truncated tetherin exhibited a dominant-negative effect and was incorporated into the liberated virions. We also found colocalization of tetherin and HBV L protein at the intracellular multivesicular body, where the budding of HBV virions takes place. In line with this, electron microscopy demonstrated that HBV virions were tethered in the lumen of the cisterna membrane under tetherin expression. Finally, knockdown of tetherin or overexpression of dominant negative tetherin attenuated the IFN-α-mediated reduction of HBV virion release. Taken together, our study suggests that IFN-α inhibits HBV virion egress from hepatocytes through the induction of tetherin. Tetherin is a host restriction factor that blocks the egress of a variety of enveloped viruses through tethering the budding virions on the cell surface with its membrane anchor domains. Here we report that interferon directly and selectively inhibits the secretion of HBV virions, but not subviral particles or nonenveloped capsids, through the induction of tetherin in hepatocyte-derived cells. The antiviral function of tetherin requires the carboxyl-terminal GPI anchor, while the GPI anchor deletion mutant exhibits dominant negative activity and attaches to liberated HBV virions. Consistent with the fact that HBV is an intracellular budding virus, microscopy analyses demonstrated that the tethering of HBV virions occurs in the intracellular cisterna and that tetherin colocalizes with HBV virions on the multivesicular body, which is the HBV virion budding site. Our study not only expands the antiviral spectrum of tetherin but also sheds light on the mechanisms of interferon-elicited anti-HBV responses.

Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection.

A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

Membrane attachment via a C-terminal glycosylphosphatidylinositol anchor is critical for conversion of PrP(C) into pathogenic PrP(Sc). Therefore the effects of the anchor on PrP structure and function need to be deciphered. Three PrP variants, including full-length PrP (residues 23-231, FL_PrP), N-terminally truncated PrP (residues 90-231, T_PrP), and PrP missing its central hydrophobic region (Δ105-125, ΔCR_PrP), were equipped with a C-terminal membrane anchor via a semisynthesis strategy. Analyses of the interactions of lipidated PrPs with phospholipid membranes demonstrated that C-terminal membrane attachment induces a different binding mode of PrP to membranes, distinct from that of non-lipidated PrPs, and influences the biochemical and conformational properties of PrPs. Additionally, fluorescence-based assays indicated pore formation by lipidated ΔCR_PrP, a variant that is known to be highly neurotoxic in transgenic mice. This finding was supported by using patch clamp electrophysiological measurements of cultured cells. These results provide new evidence for the role of the membrane anchor in PrP-lipid interactions, highlighting the importance of the N-terminal and the central hydrophobic domain in these interactions.

Equine Tetherin Blocks Retrovirus Release and Its Activity Is Antagonized by Equine Infectious Anemia Virus Envelope Protein

Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism.