Descriptive analysis of Ebola virus proteins

The virion proteins of two strains of Ebola virus were compared by SDS-polyacrylamide gel electrophoresis (PAGE) and radioimmunoprecipitation (RIP). Seven virion proteins were described; an L (180K), GP (125K), NP (104K), VP40 (40K), VP35 (35K), VP30 (30K), and VP24 (24K). The RNP complex of the virus contained the L, the NP, and VP30, with VP35 in loose association with them. The GP was the major spike protein, with VP40 and VP24 making up the remaining protein content of the multilayered envelope.     

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.     

The properties of Ebola virus proteins

The paper describes the structure and functions of Ebola virus properties. It also presents information on the role of structural (NP, VP40, VP35, GP, VP30, VP24, and L) and secreted (sGP, delta-peptide, GP1, GP(1,2delta), ssGP) proteins in the viral replication cycle and in the pathogenesis of Ebola hemorrhagic fever.     

Ebola protein analyses for the determination of genetic organization

Summary Amino-acid sequencing of the purified major nucleoprotein (NP), VP 35 and VP 40 from purified Ebola virus proved that they are the protein products of the first three genes, and that the open reading frame (ORF) of the NP begins at nucleotide 470. Because of the many unusual features of the ORFs of Ebola virus, we thought that our conclusions should be substantiated. Comparisons of in vitro-translation products to purified viral proteins were used to demonstrate conclusively that the NP, VP 35 and VP 40 were the protein products of genes one, two, and three, respectively. Studies using antibodies to synthetic peptides matching the N- and C-termini of the deduced sequences from these genes confirmed these conclusions and that the ORF for the NP begins at nucleotide 470. Subsequent studies confirmed that VP 30 is encoded by the fifth gene.     

Ultrastructural localization of L and NS enzyme subunits on vesicular stomatitis virus RNPs using gold sphere-staphylococcal protein A-monospecific IgG conjugates

Colloidal gold spheres were coated with staphylococcal protein A and were used to determine the location of NS and L proteins on vesicular stomatitis virus (VSV) ribonucleoprotein (RNP) complexes using monospecific anti-NS and anti-L IgG preparations. Conjugates using either anti-NS or anti-L demonstrated that these enzyme subunits were uniformly distributed along the entire length of the RNP complex. Under saturating conditions of IgG concentrations, it was observed that there were at least 60-70 molecules of NS protein and 30-35 molecules of L protein labeled per RNP complex.     

Assessment of the specificity of cytotoxic T lymphocytes for the nucleoprotein of Pichinde virus using recombinant vaccinia viruses

Summary Pichinde virus (PV) infection of mice results in induction of a strong H-2 restricted, virus-specific cytotoxic T lymphocyte (CTL) response and rapid clearance of the virus. To define the specificities of CTL induced by PV infection, we constructed vaccinia virus recombinants containing cloned cDNAs corresponding to full-length (VVNP) and a truncated form (VVNP_51–561) of the nucleoprotein (NP) gene of PV. Radioimmunoprecipitation analysis of infected cell lysates indicated that VVNP expressed a PV-specific product identical in size to that of authentic NP, while vaccinia virus recombinants containing truncated NP produced a polypeptide consistent with the synthesis of amino acids 51–561 of Pichinde virus NP. Interestingly, cells infected with VVNP synthesized easily detectable, but much lower levels of nucleoprotein relative to both PV and VVNP_51–561. Primary virus-specific CTL induced in three different strains of inbred mice following intravenous infection with PV were able to lyse syngeneic target cells infected with PV but did not markedly lyse syngeneic targets expressing full-length or truncated NP following recombinant vaccinia virus infection. Similarly, secondary anti-PV specific CTL generated following in vitro restimulation by PV or selectively restimulated with vaccinia recombinants did not significantly lyse target cells expressing NP. Further, infection of mice with VVNP and VVNP_51–561 did not induce CTLs specific for PV and did not prime mice for the generation of memory anti-PV CTL in vivo. These results suggest that PV gene products other than NP, such as the GPC or L protein, contain the major target epitope(s) recognized by PV-specific CTL.     

Morphogenesis and structure of caprine respiratory syncytial virus

Summary Cell cultures inoculated with caprine respiratory syncytial virus (RSV) were studied with light, fluorescent, and electron microscopy to determine the morphogenesis and structure of the virus.Small syncytia were seen after 36 hours in culture. After 48 hours in culture, syncytia were large and numerous and pleomorphic cytoplasmic inclusions were seen. These inclusions were more pronounced and numerous later in the infection cycle.Indirect immunofluorescence revealed a diffuse to granular cytoplasmic fluorescence with fluorescing fibrils on the cell surface.With the electron microscope, filamentous (100–160 nm) and spherical (90–160 nm) particles were seen budding off the cell membrane. The number of virus buds diminished with increased size of syncytia. Granular pleomorphic cytoplasmic inclusions were seen near the nucleus, and electron dense masses were seen just beneath the cytoplasmic membrane where large quantities of virus were budding from the cell surface. The first type of inclusion had distinct borders; the second diffuse borders and appeared to contain viral nucleoprotein.Negative staining revealed spherical, pleomorphic, and filamentous forms of the virus; the last form predominated. The virions were covered with club-shaped projections, and the nucleocapsids were seen as fragile strands frequently broken into fragments or as isolated rings.Morphogenesis and structure of the caprine RSV places this virus with the Pneumovirus genus of the Paramyxoviridae family.With 4 Figures     

A luciferase-based budding assay for Ebola virus

The VP40 matrix protein of Ebola virus (EBOV) is capable of budding from mammalian cells as a virus-like particle (VLP) and is the major protein involved in virus egress. A functional budding assay has been developed based upon this characteristic of VP40 to assess the contributions of VP40 sequences as well as host proteins to the budding process. This well-defined assay has been modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated into budding VP40 VLPs. Furthermore, co-expression of EBOV glycoprotein (GP) enhances release of VLPs containing VP40 and luciferase. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40, luciferase levels in the VLP fraction decrease significantly. This assay represents a promising high-throughput approach to identify inhibitors of EBOV budding.     

Immunogenicity,antigenicity, and protection efficacy of baculovirus expressed VP4 trypsin cleavage products,VP5(1)* and VP8* from rhesus rotavirus

Summary Rhesus rotavirus (RRV) VP4 trypsin cleavage product VP5(1)*, a truncated form of VP5*, was expressed in baculovirus and found by immunoprecipitation to be antigenically similar to VP5* on the virion. Immunization of mice with VP5(1)* elicited neutralizing antibody that was found to be cross-reactive with viruses representing P genotypes 1, 3, 4, 6, 7, and 8. Baculovirus expressed trypsin cleavage products, VP8* (amino acids 1–246) and VP5(1)* (amino acids 247–474), were tested for their ability to elicit a protective response in a murine model of passive protection. These results were compared to those obtained with baculovirus expressed RRV VP4. Dams immunized with baculovirus expressed RRV VP4 gave birth to pups protected from RRV virus challenge. Neither VP5(1)* nor VP8* was as effective at generating protective immunity as full length VP4. However, antibody to VP5(1)* was more effective than antibody to VP8* at mediating protection even though the neutralizing antibody titers as measured by hemagglutination inhibition and focus reduction neutralization were similar.     

Monoclonal antibodies to Marburg virus proteins and their immunochemical characteristics]

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus proteins are prepared. Positive hybridomas were selected by solid-phase enzyme immunoassay (EIA) by specificity of their immunoglobulin reaction with Marburg virus antigen. Virus proteins reacting with MAbs were identified by immunoblotting. Out of 20 examined hybridoma antibodies, 5 reacted with protein VP35, 5 with VP40, 3 with NP, 1 with protein complex VP35-VP40, MAb 7H10 detected 2 proteins (VP40 and NP), and 5 MAbs did not bind virus proteins in this assay. Marburg virus antigen adsorbed on the surface of plates were detected by indirect EIA with biotin-treated MAbs (PEIA-MAb) and indirect EIA (IEIA-MAb). The sensitivity of both methods differed with different hybridoma antibodies and was the maximum with MAb 5F1 specific to Marburg virus nucleoprotein: 5-10 and 1-2 ng/ml for the direct and indirect methods, respectively. Purified MAbs 7C4, 7D8, and 5F1 were used as antigen captures in EIA for detecting immunoglobulins to Marburg virus in a serum from convalescent after Marburg fever. The results recommend the above MAbs for use in test systems for the diagnosis of the disease and detecting virus antigen.     

Supercoiled DNA of nuclear polyhedrosis virus ofGalleria mellonella L.

Summary Supercoiled infectious DNA was isolated from nuclear polyhedrosis virus infecting great wax moth (Galleria mellonella L.). Covalently closed DNA molecules constitute approximately 10–30 per cent in our preparations. These molecules dissappear during storage. Electron micrographs of supercoiled and open circular molecules are presented. Length of the open rings is about 50–52 µ. Infectivity of different DNA forms is discussed.With 3 Figures     

Virion associated proteins of equine rhinitis B virus 1 (ERBV1): The non-structural protein 3C co-purifies with virions

Equine rhinitis B virus (ERBV), genus Erbovirus, is most closely related to the Cardiovirus genus in the family Picornaviridae. The structural proteins (VP1–4) of erboviruses are not well described, but are predicted by sequence to be 35, 29, 26 and 7 kDa. Methods for the purification of cardioviruses (polyethylene glycol, trypsin treatment) were used to characterise the structural proteins of ERBV1. Only one of the virus proteins detected was an expected molecular mass, and this 26 kDa protein was identified as VP3 by N-terminal amino acid sequencing. N-terminal sequencing of the 56 and a 29 kDa protein identified sequences consistent with VP2 and VP1 respectively, despite these being 27 kDa larger and 6 kDa smaller than predicted. Virus purified without trypsin showed proteins more consistent with masses predicted for VP1, VP2 and VP3 at 35, 29 and 26 kDa respectively. These proteins were further identified with antibodies affinity purified to recombinant VP1, VP2, VP3 produced in E. coli. Interestingly, antibodies affinity purified to the non-structural protein 3C^pro, produced in insect cells, strongly detected a 27 kDa protein in western blots of virus purified with and without trypsin treatment, suggesting the non-structural 27 kDa 3C^pro co-purifies with ERBV1 virions.     

Comparison of influenza and parainfluenza RNP properties

Summary Fowl plague virus and Sendai virus were disrupted by Tween 20 in an alcaline medium. Whereas in the case of fowl plague virus sedimentation of the ribonucleoprotein (BNP) in sucrose gradients revealed three components sedimenting at 64S, 58S, and 48S, the RNP of Sendai virus showed a sedimentation coefficient of 110S only. During buoyant density studies in CsCl the three components of fowl plague virus RNP banded at the uniform density of 1.35–1.36 g/cm³ and the RNP of Sendai virus was found at a medium density of 1.32 g/cm³.     

Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10⁸ 50% tissue culture infective dose per milliliter [TCID₅₀ · ml⁻¹]) and murine blood (EBOV concentration of 1 × 10⁷ TCID₅₀ · ml⁻¹) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.