Will we have and why do we need an Ebola vaccine?
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Animals↗
Biomedical subjects
H D Klenk
Publications and source records attributed to H D Klenk.
Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system.
We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.
Animals↗
An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.
The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.
Animals↗
Recombinant measles virus requiring an exogenous protease for activation of infectivity.
Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar(tm)-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.
Amino Acid Sequence↗
Characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants.
Transmissible gastroenteritis coronavirus (TGEV) agglutinates erythrocytes of several species by virtue of sialic acid binding activity of the surface protein S. We have isolated and characterized five haemagglutination-defective (HAD) mutants. In contrast to the parental virus, the mutants were unable to bind to porcine submandibulary mucin, a substrate rich in sialic acid. Each of the mutants was found to contain a single point mutation in the S protein (Cys155Phe, Met195Val, Arg196Ser, Asp208Asn or Leu209Pro), indicating that these amino acids are affecting the sialic acid binding site. In four of the HAD mutants a nearby antigenic site is affected in addition to the sialic acid binding site, as indicated by reactivity with monoclonal antibodies. The parental virus was found to have an increased resistance to the detergent octylglucoside compared to the HAD mutants. This effect depended on cellular sialoglycoconjugates bound to the virion. If the binding of sialylated macromolecules was prevented by neuraminidase treatment, the parental virus was as sensitive to octylglucoside as were the HAD mutants. We discuss the possibility that the sialic acid binding activity helps TGEV to resist detergent-like substances encountered during the gastrointestinal passage and thus facilitates the infection of the intestinal epithelium. An alternative function of the sialic acid binding activity - accessory binding to intestinal tissues - is also discussed.
Animals↗
Crystallization and preliminary X-ray analysis of the matrix protein from Ebola virus.
The matrix protein from Ebola virus is a membrane-associated molecule that plays a role in viral budding. Despite its functional similarity to other viral matrix proteins, it displays no sequence similarity and hence may have a distinct fold. X-ray diffraction quality crystals of the Ebola VP40 matrix protein were grown by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 64.4, b = 91.1, c = 47.9 A, beta = 96.3 degrees. A data set to 1.9 A resolution has been collected using synchrotron radiation. The unit cell contains one molecule of molecular weight 35 kDa per asymmetric unit, with a corresponding volume solvent content of 35%.
Crystallization↗
Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics.
The hemagglutinin (HA) of fowl plague virus A/FPV/Rostock/34 (H7N1) carries two N-linked oligosaccharides attached to Asn123 and Asn149 in close vicinity to the receptor-binding pocket. In previous studies in which HA mutants lacking either one (mutants G1 and G2) or both (mutant G1,2) glycosylation sites had been expressed from a simian virus 40 vector, we showed that these glycans regulate receptor binding affinity (M. Ohuchi, R. Ohuchi, A. Feldmann, and H. D. Klenk, J. Virol. 71:8377-8384, 1997). We have now investigated the effect of these mutations on virus growth using recombinant viruses generated by an RNA polymerase I-based reverse genetics system. Two reassortants of influenza virus strain A/WSN/33 were used as helper viruses to obtain two series of HA mutant viruses differing only in the neuraminidase (NA). Studies using N1 NA viruses revealed that loss of the oligosaccharide from Asn149 (mutant G2) or loss of both oligosaccharides (mutant G1,2) has a pronounced effect on virus growth in MDCK cells. Growth of virus lacking both oligosaccharides from infected cells was retarded, and virus yields in the medium were decreased about 20-fold. Likewise, there was a reduction in plaque size that was distinct with G1,2 and less pronounced with G2. These effects could be attributed to a highly impaired release of mutant progeny viruses from host cells. In contrast, with recombinant viruses containing N2 NA, these restrictions were much less apparent. N1 recombinants showed lower neuraminidase activity than N2 recombinants, indicating that N2 NA is able to partly overrule the high-affinity binding of mutant HA to the receptor. These results demonstrate that N-glycans flanking the receptor-binding site of the HA molecule are potent regulators of influenza virus growth, with the glycan at Asn149 being dominant and that at Asn123 being less effective. In addition, we show here that HA and NA activities need to be highly balanced in order to allow productive influenza virus infection.
Animals↗
Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34 (H7N1) in chicken embryos.
The tissue tropism and spread of infection of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken embryos. As shown by in situ hybridization, the virus caused generalized infection that was strictly confined to endothelial cells in all organs. Studies with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49 (H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To further analyze the factors determining endotheliotropism, the HA-activating protease furin was cloned from chicken tissue. Ubiquitous expression of furin and other proprotein convertases in the chick embryo indicated that proteolytic activation of HA was not responsible for restriction of infection to the endothelium. To determine the expression of virus receptors in embryonic tissues, histochemical analysis of alpha2,3- and alpha2,6-linked neuraminic acid was carried out by lectin-binding assays. These receptors were found on endothelial cells and on several epithelial cells, but not on tissues surrounding endothelia. Finally, we analyzed the polarity of virus maturation in endothelial cells. Studies on cultured human endothelial cells employing confocal laser scanning microscopy revealed that HA is specifically targeted to the apical surface of these cells, and electron microscopy of embryonic tissues showed that virus maturation occurs also at the luminar side. Taken together, these observations indicate that endotheliotropism of FPV in the chicken embryo is determined, on one hand, by the high cleavability of HA, which mediates virus entry into the vascular system, and, on the other hand, by restricted receptor expression and polar budding, which prevent spread of infection into tissues surrounding endothelia.
Animals↗
Identification of a novel consensus sequence at the cleavage site of the Lassa virus glycoprotein.
The Lassa virus glycoprotein consists of an amino-terminal and a carboxy-terminal cleavage fragment designated GP-1 and GP-2, respectively, that are derived by proteolysis from the precursor GP-C. The membrane-anchored GP-2 obtained from purified virions of the Josiah strain revealed the N-terminal tripeptide GTF(262) when analyzed by Edman degradation. Upstream of this site, GP-C contains the tetrapeptide sequence RRLL(259), which is conserved in all Lassa virus isolates published to date. Systematic mutational analysis of vector-expressed GP-C revealed that the motif R-X (L/I/V)-L(259) (where X stands for L, I, or V) is essential for cleavage of the peptide bond between leucine(259) and glycine(260). This cleavage motif is homologous to the consensus sequence recognized by a novel class of cellular endoproteases which have so far not been implicated in the processing of viral glycoproteins.
Amino Acid Sequence↗
Recycling of furin from the plasma membrane. Functional importance of the cytoplasmic tail sorting signals and interaction with the AP-2 adaptor medium chain subunit.
The predominant intracellular localization of the eukaryotic subtilisin-like endoprotease furin is the trans-Golgi network (TGN), but a small fraction is also found on the cell surface. Furin on the cell surface is internalized and delivered to the TGN. The identification of three endocytosis motifs, a tyrosine (YKGL(765)) motif, a leucine-isoleucine (LI(760)) motif, and a phenylalanine (Phe(790)) signal, in the furin cytoplasmic domain suggested that endocytosis of furin occurs via an AP-2/clathrin-dependent pathway. Since little is known about proteins containing multiple sorting components in their cytoplasmic domain, the combination of diverse internalization signals in the furin tail raised the question of their individual role. Here we present data showing that the furin tail interacts with the medium (micro2) subunit of the AP-2 plasma membrane-specific adaptor complex in vitro and that this interaction primarily depends on recognition of the tyrosine-based sorting signal and to less extent on the leucine-isoleucine motif. We further provide evidence that the three endocytosis signals are of different functional importance for furin internalization and retrieval to the TGN in vivo, with the tyrosine-based motif being the major determinant, followed by the phenylalanine signal, whereas the leucine-isoleucine motif is only a minor component. Finally, we report that phosphorylation of the furin tail by casein kinase II is not only important for efficient interaction with micro2 and internalization from the plasma membrane but also determines fast retrieval of the protein from the plasma membrane to the TGN.
Adaptor Protein Complex alpha Subunits↗
Delta-peptide is the carboxy-terminal cleavage fragment of the nonstructural small glycoprotein sGP of Ebola virus.
In the present study we have investigated processing and maturation of the nonstructural small glycoprotein (sGP) of Ebola virus. When sGP expressed from vaccinia virus vectors was analyzed by pulse-chase experiments using SDS-PAGE under reducing conditions, the mature form and two different precursors have been identified. First, the endoplasmic reticulum form sGP(er), full-length sGP with oligomannosidic N-glycans, was detected, sGP(er) was then replaced by the Golgi-specific precursor pre-sGP, full-length sGP containing complex N-glycans. This precursor was finally converted by proteolysis into mature sGP and a smaller cleavage fragment, Delta-peptide. Studies employing site-directed mutagenesis revealed that sGP was cleaved at a multibasic amino acid motif at positions 321 to 324 of the open reading frame. Cleavage was blocked by RVKR-chloromethyl ketone. Uncleaved pre-sGP forms a disulfide-linked homodimer and is secreted into the culture medium in the presence of the inhibitor as efficiently as proteolytically processed sGP. In vitro treatment of pre-sGP by purified recombinant furin resulted in efficient cleavage, confirming the importance of this proprotein convertase for the processing and maturation of sGP. Delta-peptide is also secreted into the culture medium and therefore represents a novel nonstructural expression product of the GP gene of Ebola virus. Both cleavage fragments contain sialic acid, but only Delta-peptide is highly O-glycosylated.
Amino Acid Sequence↗
Cloning and characterization of gp36, a human mucin-type glycoprotein preferentially expressed in vascular endothelium.
A mucin-type glycoprotein has been described in murine, rat and canine tissues as a differentiation antigen and influenza-virus receptor. We have cloned a cDNA from human placenta RNA encoding the corresponding human protein, a type-I integral membrane protein of 162 amino acids. Madin-Darby canine kidney cells transfected with the cDNA clone directed the cell-surface expression of a 36-kDa O-glycosylated sialoglycoprotein, gp36, and two minor isoforms of 28 and 70 kDa. gp36 has a broad tissue distribution with strong expression in lung, placenta and skeletal muscle, as shown by PCR screening of different cDNA libraries. Immunohistochemical detection of gp36 in cryo-sections of human placenta, kidney, lung and nasal polyps showed that the glycoprotein is expressed at the apical plasma membrane of vascular endothelial cells. Expression of gp36 was not restricted to endothelial cells, as alveolar epithelial cells were found to express gp36 as well.
Amino Acid Sequence↗
Sorting of furin at the trans-Golgi network. Interaction of the cytoplasmic tail sorting signals with AP-1 Golgi-specific assembly proteins.
The eukaryotic subtilisin-like endoprotease furin is found predominantly in the trans-Golgi network (TGN) and cycles between this compartment, the cell surface, and the endosomes. There is experimental evidence for endocytosis from the plasma membrane and transport from endosomes to the TGN, but direct exit from the TGN to endosomes via clathrin-coated vesicles has only been discussed but not directly shown so far. Here we present data showing that expression of furin promotes the first step of clathrin-coat assembly at the TGN, the recruitment of the Golgi-specific assembly protein AP-1 on Golgi membranes. Further, we report that furin indeed is present in isolated clathrin-coated vesicles. Packaging into clathrin-coated vesicles requires signal components in the furin cytoplasmic domain which can be recognized by AP-1 assembly proteins. We found that besides depending on the phosphorylation state of a casein kinase II site, interaction of the furin tail with AP-1 and its mu1subunit is mediated by a tyrosine motif and to less extent by a leucine-isoleucine signal, whereas a monophenylalanine motif is only involved in binding to the intact AP-1 complex. This study implies that high affinity interaction of AP-1 or mu1 with the cytoplasmic tail of furin needs a complex interplay of signal components rather than one distinct signal.
Amino Acid Sequence↗
The nucleoprotein of Marburg virus is target for multiple cellular kinases.
The nucleoprotein (NP) of Marburg virus is phosphorylated at serine and threonine residues in a ratio of 85:15, regardless of whether the protein is isolated from virions or from eukaryotic expression systems. Phosphotyrosine is absent. Although many potential phosphorylation sites are located in the N-terminal half of NP, this part of the protein is not phosphorylated. Analyses of phosphorylation state and phosphoamino acid content of truncated NPs expressed in HeLa cells using the vaccinia virus T7 expression system led to the identification of seven phosphorylated regions (region I*, amino acids 404-432; II*, amino acids 446-472; III*, amino acids 484-511; IV*, amino acids 534-543; V*, amino acid 549; VI*, amino acids 599-604; and VII*, amino acid 619) with a minimum of seven phosphorylated amino acid residues located in the C-terminal half of NP. All phosphothreonine residues and consensus recognition sequences for protein kinase CKII are located in regions I*-V*. Regions VI* and VII* contain only phosphoserine with three of four serine residues in consensus recognition motifs for proline-directed protein kinases. Mutagenesis of proline-adjacent serine residues to alanine or aspartic acid did not influence the function of NP in a reconstituted transcription/replication system; thus it is concluded that serine phosphorylation in the most C-terminal part of NP is not a regulatory factor in viral RNA synthesis.
Amino Acid Sequence↗
The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.
Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.
Animals↗
Strain-specific differences in the effect of influenza A virus neuraminidase on vector-expressed hemagglutinin.
In order to evaluate the efficiency of the removal of sialic acid residues from the influenza virus hemagglutinin by the viral neuraminidase in the course of the virus replication cycle, CV-1 cells expressing the hemagglutinin of H7 subtype from an SV40-based vector were superinfected with influenza virus strain A/Duck/Ukraine/63 (H3N8) or A/USSR/90/77 (H1N1). Vector-expressed hemagglutinin was immunoprecipitated from cell lysates and analyzed by polyacrylamide gel electrophoresis. The data indicate that the removal of sialic acid residues from the vector-expressed H7 hemagglutinin by N1 neuraminidase of A/USSR/90/77 virus in the course of the virus replication cycle is incomplete. The results are discussed in connection with previously published data showing that the low activity of NA in wild-type influenza virus results in incomplete removal of sialic acid residues from virion components.
Animals↗
Processing and routage of HIV glycoproteins by furin to the cell surface.
Proteolytic activation of HIV-1 and HIV-2 envelope glycoprotein precursors (gp160 and gp140, respectively) occurs at the carboxyl side of a consensus motif consisting of the highly basic amino acid sequence. We have shown previously (Hallenberger et al., 1997) and confirmed in this report, that furin and PC7 can be considered as the putative physiological enzymes involved in the proteolytic activation of the HIV-1 and HIV-2 envelope precursors. In this study, we show by cell surface biotinylation and immunoprecipitation of the cell surface associated viral glycoproteins with antibodies that the mature viral envelope glycoproteins are correctly transported to the cell. membrane. Furthermore, we show that the uncleaved forms of the glycoproteins (gp160HIV-1 and gp140HIV-2) are also highly represented at the cell surface. First, transient expression of gp160 and gp140 into CV1, a cell line known to be inefficient in the proteolytic processing of the env gene, results in the expression of gp160 and gp140 at the cell surface. Moreover, HIV-1 infection of T cells also showed that gp160 is directed to the cell surface. In addition, we show that the precursor is not incorporated in the virus particle following the budding from the cell surface. Furthermore, a gp160 mutant (deficient for three carbohydrate sites on the gp41), shown to be poorly processed with the coexpressed endoproteases, is found to be transported as an uncleaved precursor to the cell surface. In contrast to HIV envelope glycoproteins, the influenza hemagglutinin precursor (HA0), that is thought to be matured by the furin-like enzymes as well, is found to be retained within the cell and is not able to reach the cell surface. Taken together, these results show that the proteolytic maturation of the viral envelope precursors of human immunodeficiency viruses type 1 and type 2 is not a prerequisite for cell surface targeting of the HIV glycoproteins. Implications of these results for antiviral immune response are discussed.
Animals↗
A mono phenylalanine-based motif (F790) and a leucine-dependent motif (LI760) mediate internalization of furin.
The eukaryotic endoprotease furin, a member of the subtilisin-related family of prohormone convertases, is synthesized and transported within the constitutive secretory pathway to the plasma membrane, from where it recycles to the trans-Golgi network (TGN). Previous studies showed that TGN-residence and recycling are mediated by the cytoplasmic tail. Two targeting determinants have been described so far, the acidic signal CPSDSEEDEG783 containing two casein kinase II (CKII) phosphorylation sites and the internalization signal YKGL765. Refined analyses of the cytoplasmic domain of furin, which was mutagenized and tagged to the influenza hemagglutinin and to the membrane cofactor protein (CD46) as reporter molecules reveal two additional internalization determinants, a leucine-isoleucine signal, LI760, and a mono phenylalanine-based motif at F790, which functions without any specific neighboring amino acid sequence. Both signals are capable of independently mediating internalization, as has been shown previously for the tyrosine-based signal. Thus, furin internalization is mediated by at least three independent endocytosis signals.
Amino Acid Sequence↗