Ari Helenius - Academia.edu (original) (raw)
Papers by Ari Helenius
The EMBO Journal, Dec 1, 1996
Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly ... more Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexinlcalreticulin complexes.
bioRxiv (Cold Spring Harbor Laboratory), Jun 5, 2020
SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than ... more SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide 1,2. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2 (3,4). For SARS-CoV-. CC-BY-NC-ND 4.
Journal of Biological Chemistry, Sep 1, 1993
The folding of influenza hemagglutinin was analyzed after in vitro translation and translocation ... more The folding of influenza hemagglutinin was analyzed after in vitro translation and translocation into dog pancreas microsomes. Ectodomain folding of this membrane glycoprotein involves the formation of six intrachain disulfide bonds. After translation under reducing conditions, the folding process was initiated by the addition of oxidized glutathione or diamide. For correct folding a reduction-oxidation potential of -310 to -210 mV had to be reached in the bulk solution. At lower values, or after addition of other oxidants such as NAD or NADP, no HA disulfides formed. At more oxidizing values interchain disulfide-cross-linked aggregates were generated. Judging by their electrophoretic gel mobility and immunoreactivity, the folding intermediates observed in microsomes were indistinguishable from those previously seen in the endoplasmic reticulum of live cells. The kinetics of folding was also similar, but the efficiency being 43% was somewhat lower. The folding process was dependent on lumenal factors within the rough endoplasmic reticulum vesicles and also on some macromolecular component(s) present in the reticulocyte lysate. The results showed that dog pancreas microsomes provide a useful system for protein folding studies.
Journal of Virology, Dec 1, 2007
Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions.... more Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.
Current Biology, Dec 1, 1993
Biology of the Cell, 1984
The low pH dependent membrane fusion reaction responsible for the delivery of the Semliki Forest ... more The low pH dependent membrane fusion reaction responsible for the delivery of the Semliki Forest virus genome into the host cell for replication was visualized by electron microscopy. In order to increase the frequency at which fusion images could be detected a reversible inhibitor, ammonium chloride, was used to synchronize endosomal acidification, and 20 degrees C incubation was employed to concentrate virus particles into the endosomal compartment.
Lipid Absorption: Biochemical and Clinical Aspects, 1976
Deoxycholate (DOC) and other bile salts are frequently used to solubilize biomembranes. They perm... more Deoxycholate (DOC) and other bile salts are frequently used to solubilize biomembranes. They permit the isolation of membrane proteins in soluble form usually without denaturation. The mechanisms of action of bile salts are still largely unknown. In order to study the action of DOC (and other surface-active agents) we have used the Semliki Forest as a simple membrane model. This virus consists of a spherical nucleocapsid surrounded by a bilayer membrane, which is derived from the host cell plasma membrane during viral morphogenesis. The membrane contains only three polypeptide species, all specified by the viral genome.
Viruses are infectious agents that multiply by entering live cells and using the biosynthetic mac... more Viruses are infectious agents that multiply by entering live cells and using the biosynthetic machinery and other services of the host cells to produce new virus particles. Because they themselves have no metabolism, motility, or other complex functions associated with cell life, they can be extremely small and their structure can be simple. However from initial cell entry to release of progeny virus, they are entirely dependent on assistance by the host cells. To understand viruses and virus-induced diseases, cell biological understanding is of paramount importance. In this article, some basic properties of animal viruses and virus-host cell interactions are discussed.
Science, Nov 13, 2020
SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than ... more SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide1,2. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2(3,4). For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors5. Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID-19.
bioRxiv (Cold Spring Harbor Laboratory), Jun 7, 2020
The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19) is the severe... more The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1. Understanding how SARS-CoV-2 enters and spreads within human organs is crucial for developing strategies to prevent viral dissemination. For many viruses, tissue tropism is determined by the availability of virus receptors on the surface of host cells 2. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as a host receptor, yet, their tropisms differ 3-5. Here, we found that the cellular receptor neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was inhibited by a monoclonal blocking antibody against the extracellular b1b2 domain of NRP1. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial cells and in the epithelial cells facing the nasal cavity. Neuropathological analysis of human COVID-19 autopsies revealed SARS-CoV-2 infected NRP1-positive cells in the olfactory epithelium and bulb. In the olfactory bulb infection was detected particularly within NRP1-positive endothelial cells of small capillaries and medium-sized vessels. Studies in mice demonstrated, after intranasal application, NRP1-mediated transport of virus-sized particles into the central nervous system. Thus, NRP1 could explain the enhanced tropism and spreading of SARS-CoV-2. MAIN TEXT An outbreak of SARS-CoV-2 infections started in December 2019 in the Chinese province of Hubei, causing a pandemic associated with a severe acute pulmonary disease named COVID-19 (coronavirus induced disease 2019) 1. A related coronavirus, SARS-CoV, led to a much smaller outbreak in 2003, possibly because infection occurs predominantly in the lower respiratory system 6. SARS-CoV-2, in contrast, spreads rapidly through active pharyngeal viral shedding 5. Yet, uptake of both viruses is mediated by the identical cellular receptor, angiotensin-converting enzyme 2 (ACE2), present at the surface of some cells, mainly in the lower respiratory epithelium and other organs such as the kidney and the gastrointestinal tract 3,4. The reason for the extended tissue tropism of SARS-CoV-2 with primary replication in the throat is unknown. One attractive hypothesis to explain differences in pathogenicity and tropism is the presence of a polybasic furin-type cleavage site, RRAR^S, at the S1-S2 junction in the SARS-CoV-2 spike protein that is absent in SARS-CoV 7. Similar sequences are found in spike proteins of many other pathogenic human viruses, including Ebola, HIV-1 and highly virulent strains of avian influenza 7,8. Insertion of a polybasic cleavage site results in enhanced
Journal of Virology, Oct 15, 2017
The EMBO Journal, Mar 1, 1990
The influenza hemagglutinin precursor (HAO) and many other glycoproteins fold and oligomerize in ... more The influenza hemagglutinin precursor (HAO) and many other glycoproteins fold and oligomerize in the endoplasmic reticulum (ER). Only correctly folded oligomers are transported to the cell surface. To analyse the rules which determine this type of ER sorting, we have extended our analysis of hemagglutinin transport to two soluble, anchor-free recombinant HAOs derived from X31/A/Aichi/68 and A/Japan/305/57 influenza A. The results showed that individual monomers rapidly acquired a folded structure similar to that of monomeric membrane-anchored HAO. They were efficiently transported and secreted, but oligomerization was not required for secretion. Trimers or higher order complexes were either not formed (X31 HAO), or appeared during passage through the late compartments of the secretory pathway, with no effect on the rate of transport (Japan HAO). However, when initial folding was disturbed by inhibition of N-linked glycosylation, anchor-free X31 HAO was misfolded and retained in the ER as disulfide-linked complexes associated with binding protein, BiP (GRP78). The complexes were sinilar to those seen for the nonglycosylated membrane-bound HAO, but instead of forming immediately after synthesis they appeared with a half-time of 6 min. Taken together, the data demonstrate that the structural criteria that makes the anchor-free HAO transport competent are less stringent than those for the membrane form; they must fold correctly but do not need to oligomerize.
Lippincott Williams & Wilkins eBooks, 2007
Biochemistry, May 1, 1975
The dissociation of Semliki Forest virus induced by increasing concentrations of the anionic dete... more The dissociation of Semliki Forest virus induced by increasing concentrations of the anionic detergent sodium dodecyl sulfate was studied using density gradient centrifugation. Detectable binding to the virus started well below the critical micellar concentration of the detergent and increased thereafter with increased detergent concentration. At 4O there were about 11,000 binding sites per virus particle with an average association constant of about lo5 M-l. The extent of virus dissociation could be con-From the
Journal of Biological Chemistry, Nov 1, 1996
The EMBO Journal, Dec 1, 1996
Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly ... more Calnexin and calreticulin are lectin-like molecular chaperones that promote folding and assembly of newly synthesized glycoproteins in the endoplasmic reticulum. While it is well established that they interact with substrate monoglucosylated N-linked oligosaccharides, it has been proposed that they also interact with polypeptide moieties. To test this notion, glycosylated forms of bovine pancreatic ribonuclease (RNase) were translated in the presence of microsomes and their folding and association with calnexin and calreticulin were monitored. When expressed with two N-linked glycans in the presence of micromolar concentrations of deoxynojirimycin, this small soluble protein was found to bind firmly to both calnexin and calreticulin. The oligosaccharides were necessary for association, but it made no difference whether the RNase was folded or not. This indicated that unlike other chaperones, calnexin and calreticulin do not select their substrates on the basis of folding status. Moreover, enzymatic removal of the oligosaccharide chains using peptide N-glycosidase F or removal of the glucoses by ER glucosidase II resulted in dissociation of the complexes. This indicated that the lectin-like interaction, and not a protein-protein interaction, played the central role in stabilizing RNase-calnexinlcalreticulin complexes.
bioRxiv (Cold Spring Harbor Laboratory), Jun 5, 2020
SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than ... more SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide 1,2. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2 (3,4). For SARS-CoV-. CC-BY-NC-ND 4.
Journal of Biological Chemistry, Sep 1, 1993
The folding of influenza hemagglutinin was analyzed after in vitro translation and translocation ... more The folding of influenza hemagglutinin was analyzed after in vitro translation and translocation into dog pancreas microsomes. Ectodomain folding of this membrane glycoprotein involves the formation of six intrachain disulfide bonds. After translation under reducing conditions, the folding process was initiated by the addition of oxidized glutathione or diamide. For correct folding a reduction-oxidation potential of -310 to -210 mV had to be reached in the bulk solution. At lower values, or after addition of other oxidants such as NAD or NADP, no HA disulfides formed. At more oxidizing values interchain disulfide-cross-linked aggregates were generated. Judging by their electrophoretic gel mobility and immunoreactivity, the folding intermediates observed in microsomes were indistinguishable from those previously seen in the endoplasmic reticulum of live cells. The kinetics of folding was also similar, but the efficiency being 43% was somewhat lower. The folding process was dependent on lumenal factors within the rough endoplasmic reticulum vesicles and also on some macromolecular component(s) present in the reticulocyte lysate. The results showed that dog pancreas microsomes provide a useful system for protein folding studies.
Journal of Virology, Dec 1, 2007
Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions.... more Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.
Current Biology, Dec 1, 1993
Biology of the Cell, 1984
The low pH dependent membrane fusion reaction responsible for the delivery of the Semliki Forest ... more The low pH dependent membrane fusion reaction responsible for the delivery of the Semliki Forest virus genome into the host cell for replication was visualized by electron microscopy. In order to increase the frequency at which fusion images could be detected a reversible inhibitor, ammonium chloride, was used to synchronize endosomal acidification, and 20 degrees C incubation was employed to concentrate virus particles into the endosomal compartment.
Lipid Absorption: Biochemical and Clinical Aspects, 1976
Deoxycholate (DOC) and other bile salts are frequently used to solubilize biomembranes. They perm... more Deoxycholate (DOC) and other bile salts are frequently used to solubilize biomembranes. They permit the isolation of membrane proteins in soluble form usually without denaturation. The mechanisms of action of bile salts are still largely unknown. In order to study the action of DOC (and other surface-active agents) we have used the Semliki Forest as a simple membrane model. This virus consists of a spherical nucleocapsid surrounded by a bilayer membrane, which is derived from the host cell plasma membrane during viral morphogenesis. The membrane contains only three polypeptide species, all specified by the viral genome.
Viruses are infectious agents that multiply by entering live cells and using the biosynthetic mac... more Viruses are infectious agents that multiply by entering live cells and using the biosynthetic machinery and other services of the host cells to produce new virus particles. Because they themselves have no metabolism, motility, or other complex functions associated with cell life, they can be extremely small and their structure can be simple. However from initial cell entry to release of progeny virus, they are entirely dependent on assistance by the host cells. To understand viruses and virus-induced diseases, cell biological understanding is of paramount importance. In this article, some basic properties of animal viruses and virus-host cell interactions are discussed.
Science, Nov 13, 2020
SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than ... more SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease that has infected more than 6.6 million people and caused over 390,000 deaths worldwide1,2. The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2(3,4). For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors5. Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID-19.
bioRxiv (Cold Spring Harbor Laboratory), Jun 7, 2020
The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19) is the severe... more The causative agent of the current pandemic and coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 1. Understanding how SARS-CoV-2 enters and spreads within human organs is crucial for developing strategies to prevent viral dissemination. For many viruses, tissue tropism is determined by the availability of virus receptors on the surface of host cells 2. Both SARS-CoV and SARS-CoV-2 use angiotensin-converting enzyme 2 (ACE2) as a host receptor, yet, their tropisms differ 3-5. Here, we found that the cellular receptor neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was inhibited by a monoclonal blocking antibody against the extracellular b1b2 domain of NRP1. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial cells and in the epithelial cells facing the nasal cavity. Neuropathological analysis of human COVID-19 autopsies revealed SARS-CoV-2 infected NRP1-positive cells in the olfactory epithelium and bulb. In the olfactory bulb infection was detected particularly within NRP1-positive endothelial cells of small capillaries and medium-sized vessels. Studies in mice demonstrated, after intranasal application, NRP1-mediated transport of virus-sized particles into the central nervous system. Thus, NRP1 could explain the enhanced tropism and spreading of SARS-CoV-2. MAIN TEXT An outbreak of SARS-CoV-2 infections started in December 2019 in the Chinese province of Hubei, causing a pandemic associated with a severe acute pulmonary disease named COVID-19 (coronavirus induced disease 2019) 1. A related coronavirus, SARS-CoV, led to a much smaller outbreak in 2003, possibly because infection occurs predominantly in the lower respiratory system 6. SARS-CoV-2, in contrast, spreads rapidly through active pharyngeal viral shedding 5. Yet, uptake of both viruses is mediated by the identical cellular receptor, angiotensin-converting enzyme 2 (ACE2), present at the surface of some cells, mainly in the lower respiratory epithelium and other organs such as the kidney and the gastrointestinal tract 3,4. The reason for the extended tissue tropism of SARS-CoV-2 with primary replication in the throat is unknown. One attractive hypothesis to explain differences in pathogenicity and tropism is the presence of a polybasic furin-type cleavage site, RRAR^S, at the S1-S2 junction in the SARS-CoV-2 spike protein that is absent in SARS-CoV 7. Similar sequences are found in spike proteins of many other pathogenic human viruses, including Ebola, HIV-1 and highly virulent strains of avian influenza 7,8. Insertion of a polybasic cleavage site results in enhanced
Journal of Virology, Oct 15, 2017
The EMBO Journal, Mar 1, 1990
The influenza hemagglutinin precursor (HAO) and many other glycoproteins fold and oligomerize in ... more The influenza hemagglutinin precursor (HAO) and many other glycoproteins fold and oligomerize in the endoplasmic reticulum (ER). Only correctly folded oligomers are transported to the cell surface. To analyse the rules which determine this type of ER sorting, we have extended our analysis of hemagglutinin transport to two soluble, anchor-free recombinant HAOs derived from X31/A/Aichi/68 and A/Japan/305/57 influenza A. The results showed that individual monomers rapidly acquired a folded structure similar to that of monomeric membrane-anchored HAO. They were efficiently transported and secreted, but oligomerization was not required for secretion. Trimers or higher order complexes were either not formed (X31 HAO), or appeared during passage through the late compartments of the secretory pathway, with no effect on the rate of transport (Japan HAO). However, when initial folding was disturbed by inhibition of N-linked glycosylation, anchor-free X31 HAO was misfolded and retained in the ER as disulfide-linked complexes associated with binding protein, BiP (GRP78). The complexes were sinilar to those seen for the nonglycosylated membrane-bound HAO, but instead of forming immediately after synthesis they appeared with a half-time of 6 min. Taken together, the data demonstrate that the structural criteria that makes the anchor-free HAO transport competent are less stringent than those for the membrane form; they must fold correctly but do not need to oligomerize.
Lippincott Williams & Wilkins eBooks, 2007
Biochemistry, May 1, 1975
The dissociation of Semliki Forest virus induced by increasing concentrations of the anionic dete... more The dissociation of Semliki Forest virus induced by increasing concentrations of the anionic detergent sodium dodecyl sulfate was studied using density gradient centrifugation. Detectable binding to the virus started well below the critical micellar concentration of the detergent and increased thereafter with increased detergent concentration. At 4O there were about 11,000 binding sites per virus particle with an average association constant of about lo5 M-l. The extent of virus dissociation could be con-From the
Journal of Biological Chemistry, Nov 1, 1996