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Biomedical subjects

A Schneemann

Publications and source records attributed to A Schneemann.

At least 19 recordsLinked to original sources
PubMed2006

Induction of a protective immune response against viral nervous necrosis in the European sea bass Dicentrarchus labrax by using betanodavirus virus-like particles.

Betanodaviruses are causative agents of viral nervous necrosis (VNN), a devastating disease of cultured marine fish worldwide. Virus particles contain a single type of coat protein that spontaneously assembles into virus-like particles (VLPs) when expressed in a baculovirus expression system. In the present study, the immunogenicity of betanodavirus VLPs and the protection they confer against VNN in the European sea bass Dicentrarchus labrax were investigated. Enzyme-linked immunosorbent assay and seroneutralization tests performed on plasma from fish vaccinated intramuscularly with doses as low as 0.1 microg of VLPs indicated that the VLPs elicited the synthesis of specific antibetanodavirus antibodies with neutralizing activity. Moreover, fish vaccinated with VLPs were protected from challenge with live virus. Both the immune response and the protective effect against viral challenge were dose dependent. Reverse transcription-PCR data indicated that higher doses of vaccine also reduced the number of fish containing detectable quantities of betanodavirus RNA on day 30 after challenge. Taken together these data strongly support the hypothesis that VLPs obtained in the baculovirus expression system may represent an effective vaccine against VNN.

Animals↗
PubMed2004

Virus stability and protein-nucleic acid interaction as studied by high-pressure effects on nodaviruses.

In this work, we evaluate the stability, dynamics and protein-nucleic acid interaction in Flock House virus (FHV). FHV is an RNA insect virus, non-enveloped, member of the family Nodaviridae. It is composed of a bipartite single-stranded RNA genome packaged in an icosahedral capsid of 180 copies of an identical protein (alpha protein). A fundamental property of many animal viruses is the post-assembly maturation required for infectivity. FHV is constructed as a provirion, which matures to an infectious virion by cleavage of alpha protein into beta and gamma subunits. We used high pressure, temperature and chemical denaturing agents to promote perturbation of the viral capsid. These effects were monitored by spectroscopy measurements (fluorescence, light scattering and CD) and size-exclusion chromatography. The data showed that FHV was stable to pressures up to 310 MPa at room temperature. The fluorescence emission and light scattering values showed small changes that were reversible after decompression. When we combined pressure and sub-denaturing urea concentrations (1 M), the changes were more drastic, suggesting dissociation of the capsid. However, these changes were reversible after pressure release. The complete dissociation of FHV could be observed only under high urea concentrations (10 M). There were no significant changes in emission spectra up to 5 M urea. FHV also was stable when we used temperature treatments (high and low). We also compared the effects of urea and pressure on FHV wild type and cleavage-defective mutant VLPs (virus-like particles). The VLPs and authentic particles are distinguishable by protein-RNA interactions, since VLPs pack cellular RNA and native particles contain viral RNA. Our results demonstrated that native particles are more stable than VLPs to physical and chemical treatments. Our data point to the specificity of the interaction between the capsid protein and the viral RNA. This specificity is crucial to the stability of the particle, which makes this interaction an excellent target for drug development.

Capsid↗
PubMed2001

Characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus.

Betanodaviruses are causative agents of neurological disorders in several species of fish. We cloned and sequenced the RNA2 segment of two grouper viruses isolated from Epinephelus malabaricus (malabaricus grouper nervous necrosis virus, MGNNV) and Epinephelus lanceolatus (dragon grouper nervous necrosis virus, DGNNV). The sequences of the two RNAs were 99% identical and comparison with previously sequenced RNA2 segments of fish nodaviruses striped jack nervous necrosis virus, Atlantic halibut virus, sea bass encephalitis virus, and greasy grouper nervous necrosis virus (GGNNV) revealed that MGNNV and DGNNV were most closely related to GGNNV. No correlation of sequence with geographical habitat was detected. The MGNNV coat protein, the gene product of RNA2, was expressed in Sf21 cells with a recombinant baculovirus system and virus-like particles (VLPs) spontaneously formed. Two types of VLPs were observed: a slower sedimenting particle was RNase-sensitive and stain-permeable, while the faster sedimenting particle survived RNase treatment and was not stain-permeable. An image reconstruction of the latter, obtained with electron cryomicroscopy data, revealed a morphology consistent with T = 3 quasi-symmetry but with features significantly different from insect nodavirus structures at the same resolution. This assembly system allows the first biophysical comparisons of fish and insect nodavirus structure, assembly, and stability.

Amino Acid Sequence↗
PubMed2001

Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein.

Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.

Amino Acid Sequence↗
PubMed2001

Viral capsid mobility: a dynamic conduit for inactivation.

Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetyl-aziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.

Animals↗
PubMed2001

Prostanoid receptor gene expression profile in human trabecular meshwork: a quantitative real-time PCR approach.

PURPOSE: To assess the expression pattern of prostanoid receptor-encoding genes in trabecular meshwork (TM) of human donor eyes. METHODS: Disposed human donor eyes (n = 10) were obtained from the Cornea Bank, Amsterdam. The TM was dissected from the scleral tissue and homogenized in lysis buffer, and total RNA was isolated. The RNA was converted into cDNA and used as a template for noncompetitive quantitative real-time polymerase chain reaction (PCR) using green fluorescent dye to quantify the accumulation of double-stranded PCR product. Specific primers for four housekeeping genes and DP, EP(1), EP(2), EP(3,) EP(4), FP, IP, and TP receptor-encoding transcripts were developed and tested for their efficiency. RESULTS: The characterized expression profile was highly reproducible in all samples, with the EP(2) receptor-encoding transcript in the highest abundance, followed by FP, TP, IP, and EP(4) at levels that were approximately 10 to 15 times lower than that of the EP(2) subtype. DP and EP(3) were at the lowest levels, which were, on average, 45 times and 228 times lower than EP(2), respectively. CONCLUSIONS: These data show that all prostanoid receptors are expressed at different levels in human TM tissue. Because the gene expression of the EP(2) receptor is, on average, 15 times more abundant than that of the EP(4) receptor, it may be expected that the increase in flow and cAMP levels in response to the activation of the EP receptors by application of prostaglandin E(1) (PGE(1)), is primarily mediated by the EP(2) receptor. These data should be considered when designing prostanoid receptor mimetics intended to enhance the aqueous humor outflow through the TM and Schlemm's canal.

Actins↗
PubMed2000

Monitoring enzyme catalysis with mass spectrometry.

Mass spectrometry is a rapid, sensitive, and accurate quantitative approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study the proteolysis of intact viral capsid proteins, the alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl-alpha-glucopyranoside and the lipoprotein lipase-catalyzed ester hydrolysis of resorufin were examined. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry were used to examine the proteolysis of viral protein capsids, providing information about capsid dynamics and the stabilizing force of viral protein/RNA interactions. In addition, k(cat) and K(m) values of enzyme-catalyzed hydrolysis were obtained (without the use of a chromophore). These results also demonstrate the effect an unnatural substrate can have on enzyme activity. Overall, mass spectrometry provides for efficient and quantitative analysis of enzyme-catalyzed reactions, as well as the direct observation of reaction dynamics.

Animals↗
PubMed1999

Formation of an RNA heterodimer upon heating of nodavirus particles.

Flock House virus is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two positive-sense RNA molecules, which are believed to be encapsidated into a single viral particle. However, evidence to support this claim is circumstantial. Here we demonstrate that exposure of nodavirus particles to heat causes the two strands of viral RNA to form a stable complex, directly establishing that both RNAs are copackaged into one virion. The physical properties of the RNA complex, the effect of heat on the particles per se, and the possible relevance of these findings to the nodavirus life cycle are presented.

Animals↗
PubMed1999

Flow after prostaglandin E1 is mediated by receptor-coupled adenylyl cyclase in human anterior segments.

PURPOSE: To assess the effect of prostaglandin (PG) F2alpha and PGE1 on flow through the trabecular meshwork in organ preserved human anterior segments. METHODS: Isolated human anterior segments were perfused under standard conditions at a constant pressure of 10 mm Hg, while flow was continuously monitored. After a stabilization period, 6 consecutive concentrations of PGs were administered. cAMP levels were determined in the perfusate at baseline conditions and at 10(-6) M PG. RESULTS: Perfusion with concentrations ranging from 10(-10) to 10(-5) M PGE1 resulted in a dose-dependent increase in flow (P < 0.0001), reaching a plateau of a 26% increase at 10(-7) M. Perfusion with PGF2alpha or placebo (Eagle's minimum essential medium) did not influence baseline flow. cAMP produced by human anterior segments increased from 4.8+/-0.6 pmol x 30 min(-1) per anterior segment at baseline to 19.2+/-4.8 pmol x 30 min(-1) per anterior segment after perfusion with 10(-6) M PGE1 (P < 0.005). Perfusion with 10(-6) M PGF2alpha did not influence baseline cAMP production. Perfusion with 10(-5) M GDP-beta-S, an inhibitor of G protein, before and in combination with 10(-6) M PGE1 completely inhibited the increase in flow and cAMP production as observed after PGE1 alone. Perfusion with 10(-5) M GDP-beta-S alone did not affect baseline cAMP production. CONCLUSIONS: In organ preserved perfused human anterior segments, flow and cAMP production in the perfusate are not mediated by receptor-coupled adenylyl cyclase activity at baseline conditions. Perfusion with PGE1 is suggested to increase flow through the trabecular meshwork by stimulation of prostanoid EP2 receptor subtype, EP4 receptor subtype, or both, coupled to G(s) protein, inducing activation of the adenylyl cyclase catalytic unit. The results may indicate a physiological role for EP2 receptor subtype, EP4 receptor subtype, or both in the modulation of flow through the trabecular meshwork after stimulation.

Adenylyl Cyclases↗
PubMed1998

Specific encapsidation of nodavirus RNAs is mediated through the C terminus of capsid precursor protein alpha.

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.

Amino Acid Sequence↗
PubMed1998

Particle polymorphism caused by deletion of a peptide molecular switch in a quasiequivalent icosahedral virus.

The capsid of flock house virus is composed of 180 copies of a single type of coat protein which forms a T=3 icosahedral shell. High-resolution structural analysis has shown that the protein subunits, although chemically identical, form different contacts across the twofold axes of the virus particle. Subunits that are related by icosahedral twofold symmetry form flat contacts, whereas subunits that are related by quasi-twofold symmetry form bent contacts. The flat contacts are due to the presence of ordered genomic RNA and an ordered peptide arm which is inserted in the groove between the subunits and prevents them from forming the dihedral angle observed at the bent quasi-twofold contacts. We hypothesized that by deleting the residues that constitute the ordered peptide arm, formation of flat contacts should be impossible and therefore result in assembly of particles with only bent contacts. Such particles would have T=1 symmetry. To test this hypothesis we generated two deletion mutants in which either 50 or 31 residues were eliminated from the N terminus of the coat protein. We found that in the absence of residues 1 to 50, assembly was completely inhibited, presumably because the mutation removed a cluster of positively charged amino acids required for neutralization of encapsidated RNA. When the deletion was restricted to residues 1 to 31, assembly occurred, but the products were highly heterogeneous. Small bacilliform-like structures and irregular structures as well as wild-type-like T=3 particles were detected. The anticipated T=1 particles, on the other hand, were not observed. We conclude that residues 20 to 30 are not critical for formation of flat protein contacts and formation of T=3 particles. However, the N terminus of the coat protein appears to play an essential role in regulating assembly such that only one product, T=3 particles, is synthesized.

Amino Acid Sequence↗
PubMed1995

The remarkable coding strategy of borna disease virus: a new member of the nonsegmented negative strand RNA viruses.

BDV uses a remarkably broad range of mechanisms to direct expression of its 8.9-kb genome. Although much remains to be elucidated, it is clear that BDV genome expression is modulated by the use of multiple strategies, including differential gene transcription, post-transcriptional modification, and translational efficiency. Further insights into the details of this multilevel system will be essential to understanding BDV biology, pathogenesis, and neurotropism.

Animals↗
PubMed1995

Borna disease virus: implications for human neuropsychiatric illness.

The cause of Borna disease, a neurological syndrome affecting mammals and birds, has recently been shown to be infection with an RNA virus. Molecular genetic analysis suggests that Borna disease virus represents a new viral taxon. It has a wide host range and is tropic for specific circuits in the central nervous system. There is indirect evidence that links it to diseases of the human central nervous system.

Animals↗
PubMed1995

The atypical strategies used for gene expression of Borna disease virus, a nonsegmented, negative-strand RNA virus.

Borna disease virus (BDV) is a neurotropic agent that causes disturbances in movement and behavior in vertebrate host species ranging from birds to primates. Although the virus has not been isolated from human subjects, there is indirect evidence to suggest that humans with neuropsychiatric disorders may be infected with BDV. Recently, virus particles have been isolated and the viral genomic RNA has been cloned. This analysis revealed that BDV is a nonsegmented, negative-strand RNA virus. Unusual features such as RNA splicing, overlap of transcription units and transcription signals, as well as sequence dissimilarity for four of five major open reading frames to genes of other nonsegmented, negative-strand RNA viruses suggest that BDV is likely to represent a new taxon within the order Mononegavirales.

Animals↗
PubMed1994

Production and crystallization of virus-like particles assembled in a heterologous protein expression system.

It is of considerable interest to separate the processes of viral infectivity and virion assembly. Until recently this has only been possible with viruses that could be disassembled and reassembled in vitro. Even in these cases it was difficult to establish the authenticity of reassembled capsid protein because of possible irreversible damage that may have occurred to the protein during disassembly. An ideal method for the study of virus assembly is a protein expression system in which conditions are appropriate for spontaneous particle formation from freshly synthesized polypeptides. The baculovirus expression system has proven to be an excellent means to this end. Recently, this approach has been used to study the T = 3 Flock House insect virus and it has been demonstrated that subunits with the wild-type protein sequence, and with site-specific mutations that prevent particle maturation, will assemble and crystallize. This same approach has now been used at Purdue to study the T = 4 Nudaurelia omega capensis insect virus. There is no cell culture system currently available for the study of NomegaV, thus the expression system provides the first opportunity to study assembly under controlled conditions.

Journal Article↗
PubMed1994

Genomic organization of Borna disease virus.

Borna disease virus is a neurotropic negative-strand RNA virus that infects a wide range of vertebrate hosts, causing disturbances in movement and behavior. We have cloned and sequenced the 8910-nucleotide viral genome by using RNA from Borna disease virus particles. The viral genome has complementary 3' and 5' termini and contains antisense information for five open reading frames. Homology to Filoviridae, Paramyxoviridae, and Rhabdoviridae is found in both cistronic and extracistronic regions. Northern analysis indicates that the virus transcribes mono- and polycistronic RNAs and uses termination/polyadenylylation signals reminiscent of those observed in other negative-strand RNA viruses. Borna disease virus is likely to represent a previously unrecognized genus, bornaviruses, or family, Bornaviridae, within the order Mononegavirales.

Amino Acid Sequence↗