Development of a highly sensitive, field operable biosensor for serological studies of Ebola virus in central Africa.

We describe herein a newly developed optical immunosensor for detection of antibodies directed against antigens of the Ebola virus strains Zaire and Sudan. We employed a photo immobilization methodology based on a photoactivatable electrogenerated poly(pyrrole-benzophenone) film deposited upon an indium tin oxide (ITO) modified conductive surface fiber-optic. It was then linked to a biological receptor, Ebola virus antigen in this case, on the fiber tip through a light driven reaction. The photochemically modified optical fibers were tested as an immunosensor for detection of antibodies against Ebola virus, in animal and human sera, by use of a coupled chemiluminescent reaction. The immunosensor was tested for sensitivity, specificity, and compared to standard chemiluminescent ELISA under the same conditions. The analyte, anti-Ebola IgG, was detected at a low titer of 1:960,000 and 1:1,000,000 for subtypes Zaire and Sudan, respectively. While the same serum tested by ELISA was one order (24 times) less sensitive.

Phosphorylation of Marburg virus VP30 at serines 40 and 42 is critical for its interaction with NP inclusions.

The Marburg virus (MBGV) nucleocapsid complex is composed of four viral proteins (NP, L, VP35, and VP30) and the negative-strand nonsegmented genomic RNA. NP, L, and VP35 are functionally conserved among the order Mononegavirales, whereas VP30, a phosphoprotein, represents a filovirus-specific nucleocapsid protein. In the present paper, we have characterized the localization and function of VP30 phosphorylation. The main phosphorylation sites are represented by seven serine residues in the region of amino acid 40 to 51 of VP30. Additionally, trace amounts of phosphothreonine were detected. Substitution of serine residues 40 and 42 by alanine abolished the interaction of VP30 with NP-induced inclusion bodies, which contain nucleocapsid-like structures formed by NP. Substitution of the other phosphoserine residues had little effect on this interaction. Replacement of the introduced alanine residues 40 and 42 by aspartate restored the interaction between VP30 and the NP inclusions pointing to the importance of negative charges at these particular positions.

Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

Adverse effects of MVA-T7 on the transport of Marburg virus glycoprotein.

Expression of glycoproteins has been carried out successfully using recombinant vaccinia virus vectors. Especially attractive is the use of recombinant vaccinia viruses which express the DNA-dependent RNA polymerase of the phage T7 (T7-polymerase). The T7-polynerase drives the transcription of plasmid-based genes under the control of the T7 RNA polymerase promoter transfected into the infected cell. Comparison of two different recombinant vaccinia viruses, vTF7-3 and MVA-T7, revealed that post-translational processing of Marburg virus surface glycoprotein (GP) is impaired in the MVA-T7 but not in the vTF7-3 system. Influenza virus hemagglutinin, however, was transported and processed like the authentic protein in both systems. It is shown that transport of GP in the MVA-T7 system is not completely blocked, but the vast majority of molecules remained Endo H-sensitive. Only trace amounts evaded the endoplasmatic reticulum and reached the plasma membrane. Thus, the adverse effects of MVA-T7 on the processing of recombinant glycoproteins cannot be predicted, and correct processing has to be investigated for every expressed glycoprotein.

Sorting of Marburg virus surface protein and virus release take place at opposite surfaces of infected polarized epithelial cells.

Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein.

The Ebola virus VP35 protein functions as a type I IFN antagonist.

An assay has been developed that allows the identification of molecules that function as type I IFN antagonists. Using this assay, we have identified an Ebola virus-encoded inhibitor of the type I IFN response, the Ebola virus VP35 protein. The assay relies on the properties of an influenza virus mutant, influenza delNS1 virus, which lacks the NS1 ORF and, therefore, does not produce the NS1 protein. When cells are infected with influenza delNS1 virus, large amounts of type I IFN are produced. As a consequence, influenza delNS1 virus replicates poorly. However, high-efficiency transient transfection of a plasmid encoding a protein that interferes with type I IFN-induced antiviral functions, such as the influenza A virus NS1 protein or the herpes simplex virus protein ICP34.5, rescues growth of influenza delNS1 virus. When plasmids expressing individual Ebola virus proteins were transfected into Madin Darby canine kidney cells, the Ebola virus VP35 protein enhanced influenza delNS1 virus growth more than 100-fold. VP35 subsequently was shown to block double-stranded RNA- and virus-mediated induction of an IFN-stimulated response element reporter gene and to block double-stranded RNA- and virus-mediated induction of the IFN-beta promoter. The Ebola virus VP35 therefore is likely to inhibit induction of type I IFN in Ebola virus-infected cells and may be an important determinant of Ebola virus virulence in vivo.

Ultrastructural organization of recombinant Marburg virus nucleoprotein: comparison with Marburg virus inclusions.

HeLa cells expressing the recombinant Marburg virus (MBGV) nucleoprotein (NP) have been studied by immunoelectron microscopy. It was found that MBGV NPs assembled into large aggregates which were in close association with membranes of the rough endoplasmic reticulum. Further analysis of these aggregates revealed that NPs formed tubule-like structures which were arranged in a hexagonal pattern. A similar pattern of preformed nucleocapsids was detected in intracellular inclusions induced by MBGV infection. Our data indicated that MBGV NP is able to form nucleocapsid-like structures in the absence of the authentic viral genome and other nucleocapsid-associated proteins.

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.

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.

Comparison of the transcription and replication strategies of marburg virus and Ebola virus by using artificial replication systems.

The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses.

Interactions of Marburg virus nucleocapsid proteins.

In this study, the components of Marburg virus nucleocapsid complex were determined, and interactions between the compounds were investigated. Using salt dissociation of isolated virions, four proteins (NP, VP35, VP30, and L) remained attached to the core complex. Same proteins were detected intracellularly to be localized in MBGV-induced inclusion bodies, which are presumed to represent areas of nucleocapsid formation. To investigate interactions between the four proteins, immunofluorescence analysis of coexpressed proteins was carried out. Complexes between NP-VP35 and NP-VP30 were formed, which was demonstrated by redistribution of VP35 and VP30 into NP-induced inclusion bodies. Furthermore, complexes between L and VP35 were detected by coimmunoprecipitation. Using deletion mutants of L, the binding site of VP35 on L could be restricted to the N-terminal 530 amino-acid residues. Coexpression of NP, VP35, and L led to the formation of a triple complex where VP35 linked NP and L. The detected complexes are presumed to represent the key components of the MBGV transcription and replication machinery.

Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virus-specific monocistronic minigenomes.

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication.

Intracellular transport and processing of the Marburg virus surface protein in vertebrate and insect cells.

The surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oliosaccharides leading to a mature protein of 170-200 kDa approximately 30 min after pulse labelling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slighly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein.

Termini of all mRNA species of Marburg virus: sequence and secondary structure.

The 3' and 5' ends of Marburg virus (MBG)-specific mRNA species have been determined using reverse transcription-PCR, rapid amplification of cDNA ends, or the reverse ligation-mediated PCR procedure after removal of cap structures with tobacco acid pyrophosphatase. The polyadenylation sites of all MBG-specific mRNAs were strictly conserved and corresponded to the predicted transcriptional stop signals of genomic RNA. Determination of the 5' ends of the mRNA species showed that mRNA synthesis started precisely at the first nucleotide of a highly conserved transcriptional start site. The 5' ends of the mRNA species can build a stable secondary structure with the conserved nucleotides always located in the stem region of a hairpin. Nucleotide substitutions in the conserved 5' regions are accompanied by compensatory mutations of the complementary nucleotide thus leading to a conservation of the secondary structures. Compensatory mutations were also found when 5' ends of mRNA of MBG strain Musoke were compared with MBG strain Popp or the closely related Ebola virus, indicating that the secondary structures will be conserved even if the sequence is altered.

Acylation of the Marburg virus glycoprotein.

The surface protein of Marburg virus (GP) is modified by acylation, as shown by labeling with [3H]myristic and [3H]palmitic acid. Acylation of GP also occurred when it was expressed in insect cells with the baculovirus expression system. Gas chromatographic analyses of the bound fatty acids indicated that exogenously added [3H]myristic acid was partly metabolized to palmitic and stearic acid. To elucidate the nature of the fatty acid bond, [3H]palmitic acid-labeled GP was treated with mercaptoethanol. Since the fatty acids were removed by this treatment, it is concluded that the linkage is of the thioester type. A putative attachment site for thioester-linked fatty acids consisting of two cysteine residues located between the transmembrane anchor and the carboxy-terminal cytoplasmic tail of GP (Cys671 and Cys673) could be identified. Site-directed mutagenesis of these two amino acids to alanine residues clearly demonstrated that both cysteines could serve as acylation sites.

Marburg virus gene 4 encodes the virion membrane protein, a type I transmembrane glycoprotein.

Gene 4 of Marburg virus, strain Musoke, was subjected to nucleotide sequence analysis. It is 2,844 nucleotides long and extends from genome position 5821 to position 8665 (EMBL Data Library, emnew: MVREPCYC [accession no. Z12132]). The gene is flanked by transcriptional signal sequences (start signal, 3'-UACUUCUUGUAAUU-5'; termination signal, 3'-UAAUUCUUUUU-5') which are conserved in all Marburg virus genes. The major open reading frame encodes a polypeptide of 681 amino acids (M(r), 74,797). After in vitro transcription and translation, as well as expression in Escherichia coli, this protein was identified by its immunoreactivity with specific antisera as the unglycosylated form of the viral membrane glycoprotein (GP). The GP is characterized by the following four different domains: (i) a hydrophobic signal peptide at the amino terminus (1 to 18), (ii) a predominantly hydrophilic external domain (19 to 643), (iii) a hydrophobic transmembrane anchor (644 to 673), and (iv) a small hydrophilic cytoplasmic tail at the carboxy terminus (674 to 681). Amino acid analysis indicated that the signal peptide is removed from the mature GP. The GP therefore has the structural features of a type I transmembrane glycoprotein. The external domain of the protein has 19 N-glycosylation sites and several clusters of hydroxyamino acids and proline residues that are likely to be the attachment sites for about 30 O-glycosidic carbohydrate chains. The region extending from positions 585 to 610 shows significant homology to a domain observed in the envelope proteins of several retroviruses and Ebola virus that has been suspected to be responsible for immunosuppressive properties of these viruses. A second open reading frame of gene 4 has the coding capacity for an unidentified polypeptide 112 amino acids long.

Marburg virus, a filovirus: messenger RNAs, gene order, and regulatory elements of the replication cycle.

The genome of Marburg virus (MBG), a filovirus, is 19.1 kb in length and thus the largest one found with negative-strand RNA viruses. The gene order - 3' untranslated region-NP-VP35-VP40-GP-VP30-VP24-L-5' untranslated region-resembles that of other non-segmented negative-strand (NNS) RNA viruses. Six species of polyadenylated subgenomic RNAs, isolated from MBG-infected cells, are complementary to the negative-strand RNA genome. They can be translated in vitro into the known structural proteins NP, GP (non-glycosylated form), VP40, VP35, VP30 and VP24. At the gene boundaries conserved transcriptional start (3'-NNCUNCNUNUAAUU-5') and stop signals (3'-UAAUUCUUUUU-5') are located containing the highly conserved pentamer 3'-UAAUU-5'. Comparison with other NNS RNA viruses shows conservation primarily in the termination signals, whereas the start signals are more variable. The intergenic regions vary in length and nucleotide composition. All genes have relatively long 3' and 5' end non-coding regions. The putative 3' and 5' leader RNA sequences of the MBG genome resemble those of other NNS RNA viruses in length, conservation at the 3' and 5' ends, and in being complementary at their extremities. The data support the concept of a common taxonomic order Mononegavirales comprising the Filoviridae, Paramyxoviridae, and Rhabdoviridae families.

The nucleotide sequence of the L gene of Marburg virus, a filovirus: homologies with paramyxoviruses and rhabdoviruses.

The nucleotide sequence of the L gene of Marburg virus, strain Musoke, has been determined. The L gene has a single long open reading frame encoding a polypeptide of 2330 amino acids (MW 267,175) that represents the viral RNA-dependent RNA polymerase. The putative transcription start signal (3'CUACCUAUAAUU 5') and the termination signal (3' UAAUUCUUUUU 5') of the gene could be identified. Computer-assisted comparison of the L protein with L proteins of other nonsegmented negative-stranded RNA viruses (Paramyxoviridae: Sendai virus, Newcastle disease virus, human parainfluenza 3 virus, measles virus, human respiratory syncytial virus; Rhabdoviridae: vesicular stomatitis virus, rabies virus) revealed significant homologies primarily in the N-terminal half of the proteins. We have identified three common conserved boxes (A, B, and C) among filo-, paramyxo-, and rhabdovirus L proteins, which are probably involved in the polymerase function. The L proteins can be divided into an N-terminal half, which seems to accommodate the common enzymatic sites, and a C-terminal half carrying virus specific peculiarities. The data presented here suggest a common evolutionary history for all nonsegmented negative-stranded RNA viruses and show that filoviruses are more closely related to paramyxo- than to rhabdoviruses.