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Biomedical subjects

B N Fields

Publications and source records attributed to B N Fields.

At least 73 records · Page 4Linked to original sources
PubMed1988

Uptake of reovirus serotype 1 by the lungs from the bloodstream is mediated by the viral hemagglutinin.

We used the mammalian reoviruses to determine the molecular basis of the clearance of a virus from the bloodstream by specific organs. Reovirus serotypes 1 (T1) and 3 (T3) were radiolabeled with [35S]methionine or 125I, and the viruses were injected intravenously into weanling rats. The distribution of radioactivity within the animals was determined at various times after the injection. Both viruses were cleared rapidly from the bloodstream and concentrated in different organs. Reovirus T1 was found predominantly in the lungs and liver, whereas T3 was found predominantly in the liver, with very little virus in the lungs. Using intertypic reassortants, we determined that the T1 S1 gene, which encodes the viral hemagglutinin (sigma 1 protein), is responsible for the difference in uptake of T1 and T3 by the lungs. The genetic mapping was extended by using several approaches. (i) T1 subjected to limited proteolytic digestion with chymotrypsin was cleared efficiently by the lungs despite the removal of sigma 3 and digestion of mu 1C to delta. (ii) Uptake of T1 by the lungs was totally inhibited by incubation of T1 with an anti-sigma 1 monoclonal antibody or its Fab fragment before injection. (iii) A reovirus T1 variant in the sigma 1 protein was poorly taken up by the lungs. These data indicate that clearance of reovirus from the bloodstream by the lungs is dependent on the presence of the T1 sigma 1 protein.

Animals↗
PubMed1988

Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.

By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.

Amino Acid Sequence↗
PubMed1987

Powerful prions?

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Central Nervous System Diseases↗
PubMed1987

Visualization of viral clearance in the living animal.

The early events in viral dissemination via the bloodstream were identified by monitoring the fate of 123I-radiolabeled reovirus after it was injected intravenously in rats. Continuous scintillation camera imaging showed that reovirus serotypes 1 and 3 were cleared from the circulation in less than 10 minutes by specific and distinct target organs. Reovirus serotype 1 accumulated predominantly in the lungs and the liver, whereas serotype 3 accumulated in the liver and the spleen with very little virus uptake by the lungs. Incubation of reovirus serotype 1 with a monoclonal antibody directed against the viral hemagglutinin before injection totally inhibited the clearance of the virus by the lungs. Similar results were obtained when viruses biolabeled with 35S were used. These results demonstrate that viruses can be rapidly transported through the bloodstream to specific target organs and that the localization of the viruses depends on the interaction between specific viral surface components and the target organ.

Animals↗
PubMed1987

Expression of reovirus type 3 (Dearing) sigma 1 and sigma s polypeptides in Escherichia coli.

The reovirus S1 gene codes for two polypeptides: sigma 1 and sigma s. In order to characterize the structure and function of the sigma 1 polypeptide, we have expressed the sigma 1 protein in Escherichia coli. The S1 gene from mammalian reovirus type 3 (Dearing strain) and the variant K strain were subcloned into an expression vector containing the tac (trp-lac) promoter designed to express foreign gene products in E. coli efficiently. The hybrid plasmids, upon induction with isopropyl-beta-D-thiogalactopyranoside, expressed two polypeptides that were detected by [35S]methionine labelling. One of the induced proteins had a relative molecular mass (Mr) of approx. 46,000 and corresponded to sigma 1, as shown by immunoprecipitation with goat anti-reovirus antibody and a monoclonal antibody against sigma 1. The second induced protein had a Mr of approx. 12,000 and was very similar to sigma s as judged by comparative tryptic peptide map analysis. Protein sigma 1 produced in E. coli was shown to be functional as judged from its ability to bind to mouse L fibroblasts.

Animals↗
PubMed1987

Evidence that the sigma 1 protein of reovirus serotype 3 is a multimer.

In this report, we study the reovirus serotype 3 (strain Dearing) sigma 1 protein obtained from various sources: from Escherichia coli expressing sigma 1 protein, from reovirus-infected mouse L cells, and from purified reovirions. We demonstrate that the sigma 1 protein is a multimer in its undisrupted form and present biochemical evidence suggesting that the multimer is made up of four sigma 1 subunits.

Animals↗
PubMed1987

Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle.

Lysosomotropic drugs such as NH4Cl have been useful for studying the role of low pH in early events in virus infection. NH4Cl blocks the production of infectious progeny virus in mammalian reovirus-infected cells. The inhibitory effect of NH4Cl is mediated by an inhibition of intracellular digestion of reovirus outer capsid proteins. In vitro digestion of viral outer capsid proteins produces infectious partially uncoated particles, called intermediate subviral particles, which are no longer inhibited by the presence of NH4Cl. These results indicate that proteolytic processing of reovirus outer capsid proteins takes place in a low pH compartment of the cell and is an essential step in the viral infectious cycle.

Ammonium Chloride↗
PubMed1986

Syngeneic monoclonal internal image anti-idiotopes as prophylactic vaccines.

A syngeneic monoclonal anti-idiotope that behaves as an internal image of the mammalian reovirus type 3 cellular attachment protein (viral hemagglutinin) was used in the syngeneic host for the induction of a prophylactic anti-viral antibody response. These studies were performed without the aid of co-stimulation by viral antigens. The high stringency of this system enables us to define the maximum constraints on the use of anti-idiotopes as anti-viral vaccines. We have used the murine BALB/c monoclonal IgM anti-idiotope 87.92.6 to study the idiotope and antigen specificity, kinetics, dose dependence, adjuvant, carrier, and valency requirements of anti-idiotope-induced anti-viral antibody responses. These studies show that the production of high titer neutralizing antibody requires a lengthy (60 day) immunization protocol, which includes the use of adjuvant and multivalent anti-idiotope, and is dependent on anti-idiotope concentrations of greater than 50 micrograms. When administered in this manner anti-idiotope can stimulate serotype-specific antibody responses across species barriers at levels comparable with those obtained after inoculation with virus. The practical efficacy of these reagents and procedures is documented by the ability of maternal immunization with anti-idiotope to confer complete protection in neonates from a potentially lethal reovirus type 3 viral infection.

Animals↗
PubMed1986

Distinct pathways of viral spread in the host determined by reovirus S1 gene segment.

The genetic and molecular mechanisms that determine the capacity of a virus to utilize distinct pathways of spread in an infected host were examined by using reoviruses. Both reovirus type 1 and reovirus type 3 spread to the spinal cord following inoculation into the hindlimb or forelimb footpad of newborn mice. For type 3 this spread is through nerves and occurs via the microtubule-associated system of fast axonal transport. By contrast, type 1 spreads to the spinal cord through the bloodstream. With the use of reassortant viruses containing various combinations of double-stranded RNA segments (genes) derived from type 1 and type 3, the viral S1 double-stranded RNA segment was shown to be responsible for determining the capacity of reoviruses to spread to the central nervous system through these distinct pathways.

Animals↗
PubMed1986

Viral shedding and transmission between hosts determined by reovirus L2 gene.

Two reovirus isolates (type 1 Lang and type 3 Dearing) differ in their transmissibility between littermates of newborn mice. They also differ in the amounts of virus excreted by the gastrointestinal tract. With the use of reassortant viruses, these properties were mapped to the L2 gene. Thus environmental spread of reovirus is a genetic property.

Animals↗
PubMed1986

Antiidiotypic antibody to reovirus binds to neurons and protects from viral infection.

A syngeneic monoclonal antiidiotype directed against the idiotype of an antireovirus type 3 hemagglutinin demonstrates several of the biological actions of the original viral hemagglutinin and binds to rat and murine cortical neurons grown in dissociated cell culture. Receptor-bearing neurons appear within 24 hours of plating in cultures from mouse or rat cortex taken on embryonic day 15; these neurons are demonstrable for the duration of the culture life span (4 to 8 weeks). When cortical cultures are incubated with antiidiotype before or during exposure to reovirus, the antiidiotype protects neurons from type 3 infection without inhibiting infection of nonneuronal cells with either type 3 or type 1. Thus an antibody directed against a viral receptor can prevent infection of receptor-bearing cells without directly neutralizing the virus.

Animals↗
PubMed1986

Nucleic acid sequence of an internal image-bearing monoclonal anti-idiotype and its comparison to the sequence of the external antigen.

The monoclonal anti-idiotypic antibody (mAb2) 87.92.6 directed against the 9B.G5 antibody specific for the virus neutralizing epitope on the mammalian reovirus type 3 hemagglutinin was previously demonstrated to express an internal image of the receptor binding epitope of the reovirus type 3. Furthermore, this mAb2 has autoimmune reactivity to the cell surface receptor of the reovirus. The nucleotide and deduced amino acid sequences of the 87.92.6 mAb2 heavy and light chains are described in this report. The sequence analysis reveals that the same heavy chain variable and joining (VH and JH) gene segments are used by the 87.92.6 anti-idiotypic mAb2 and by the dominant idiotypes of the BALB/c anti-GAT (cGAT) and anti-NP (NPa) responses. [GAT; random polymer that is 60% glutamic acid, 30% alanine, and 10% tyrosine. NP; (4-hydroxy-3-nitrophenyl)-acetyl.] Despite extensive homology at the level of the heavy chain variable regions, the NPa positive BALB/c anti-NP monoclonal antibody 17.2.25 binds neither 9B.G5 nor the cellular receptor for the hemagglutinin. Amino acid sequence comparison between the viral hemagglutinin and the 87.92.6 mAb2 light chain "internal image," reveals an area of significant homology indicating that antigen mimicry by antibodies may be achieved by sharing primary structure.

Amino Acid Sequence↗
PubMed1986

Genetic basis for altered pathogenesis of an immune-selected antigenic variant of reovirus type 3 (Dearing).

In this paper we provide a step by step comparison of the pathogenesis of murine infection caused by reovirus type 3 (Dearing) and an antigenic variant (K) selected by its resistance to neutralization with a monoclonal antibody (G5) directed against the T3 hemagglutinin. To show that specific changes in the biologic properties of variant K were due to mutation in the S1 double-stranded RNA segment (gene), which encodes the viral hemagglutinin, we generated a reassortant virus ("1 HA K") containing the variant K S1 gene and compared its properties to variant K and to a reassortant ("1 HA 3") containing the T3 (Dearing) S1 gene. These studies, in conjunction with our previous nucleotide sequence analysis of the S1 genes of variant K and T3 (Dearing) [R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizel, Jr., and B. N. Fields, Nature (London) 315:421-423, 1985; R. Bassel-Duby, D. R. Spriggs, K. L. Tyler, and B. N. Fields, submitted for publication], indicate that a single amino acid change in the T3 hemagglutinin can alter viral growth and tropism within the central nervous system without affecting either its primary replication in the intestine or its pattern of spread to or within the central nervous system.

Animals↗
PubMed1986

Cell receptors for the mammalian reovirus: reovirus-specific T-cell hybridomas can become persistently infected and undergo autoimmune stimulation.

We have previously described the development of virus-specific helper T cell hybridomas which recognize structural determinants shared by type 1 and type 3 reoviruses that have been exposed to UV radiation. We have found that T-cell hybridomas become persistently infected with live type 3 reovirus used for the immunization. Persistently infected T-hybridoma cells were found to spontaneously produce interleukin 2 (IL-2). To analyze the mechanism of induction of IL-2 secretion of persistently infected T-cell hybridomas, we exposed T-cell hybridomas specific for UV-treated virus to replicating type 3 reovirus. The T-cell hybridomas became infected but did not produce IL-2 unless simultaneously exposed to syngeneic I-A+ antigen-presenting cells. In this situation, the persistently infected T-cell hybridomas produced IL-2 without being reexposed to virus. This process was not a consequence of nonspecific IL-2 gene activation, which occurs in cells persistently infected with reovirus, because reovirus infection did not activate IL-2 secretion in T-cell hybridomas with other antigenic specificities. Reovirus exposure also resulted in persistent infection of certain antigen-presenting B-cell tumor lines. The persistently infected B-cell tumor lines could stimulate reovirus-specific helper T cells but not T-cell hybridomas of other specificities. The data support the thesis that persistent infection of reovirus-specific T cells creates a mechanism in which the virus released from these cells is processed and then reexpressed by I-A+ antigen-presenting cells. The IA antigen and reovirus structures on the antigen-presenting cells then restimulate the T cells through their specific receptors, resulting in IL-2 synthesis and release. These observations may be relevant to mechanisms of autoimmunity induced by virus.

Animals↗
PubMed1986

Identification of attenuating mutations on the reovirus type 3 S1 double-stranded RNA segment with a rapid sequencing technique.

Reovirus type 3 variants with mutations in the major neutralization domain of the sigma 1 protein have attenuated neurovirulence and restricted neurotropism. We devised a variation of the rapid RNA sequencing technique to facilitate the analysis of double-stranded RNA. We sequenced the S1 double-stranded RNA segment, which encodes the sigma 1 protein, of five attenuated reovirus type 3 variants. Four of the variants have changes in codon 419, and a fifth variant has a change at codon 340, all of which resulted in amino acid substitutions in the sigma 1 protein. We identified two sites on the reovirus type 3 sigma 1 protein that play a critical role in neurovirulence.

Amino Acid Sequence↗
PubMed1985

Structural similarities between the mammalian beta-adrenergic and reovirus type 3 receptors.

The mechanism by which viruses bind to and infect specific tissues to cause disease has only recently begun to be understood. The mammalian reoviruses provide an especially attractive model for studying the details of cell surface recognition. The cell and tissue tropism of reovirus is determined by a portion of the viral hemagglutinin termed the neutralization domain. We have reported previously on the generation of both monoclonal and polyclonal anti-idiotypic antibodies that mimic the viral hemagglutinin in the specificity of binding to the reovirus receptor. By using these anti-idiotypic antibodies as specific probes, we have successfully isolated the mammalian reovirus receptor from neuronal and lymphoid cells. In the present study, we report that the reovirus receptor is structurally similar to the mammalian beta-adrenergic receptor. This conclusion is based on the following observations: (i) purified beta-adrenergic receptor is immunoprecipitable by anti-reovirus receptor antibody; (ii) purified reovirus receptor obtained from murine thymoma cells and beta-adrenergic receptor obtained from calf lung exhibit identical molecular masses and isoelectric points; (iii) trypsin digests of purified reovirus and beta-adrenergic receptors display indistinguishable fragment patterns; (iv) purified reovirus receptor binds the beta-antagonist [125I]iodohydroxybenzylpindolol and this binding is blocked by the beta-agonist isoproterenol.

Animals↗
PubMed1985

Isolation and biochemical characterization of the mammalian reovirus type 3 cell-surface receptor.

A cell-surface receptor for the mammalian reovirus type 3 hemagglutinin was isolated by using antiidiotypic anti-receptor antibodies. The receptor is a glycoprotein with a molecular mass of 67,000 daltons and a pI of 5.9. Evidence that the isolated structure represents the reovirus receptor was obtained by electrophoretic immunoblot studies, which demonstrated that the 67,000-dalton glycoprotein is the only cell-surface structure recognized by both reovirus type 3 and the anti-receptor immunoglobulin. Comparison of the reovirus receptor on murine thymoma (R1.1) and rat neuroblastoma (B104) cells indicated that similar structures on the cell surface are recognized by the reovirus type 3 and the anti-receptor antibodies as previously suggested from cellular and binding studies. This receptor was found on mouse, rat, monkey, and human cells. Furthermore, diverse tissue types, including lymphoid and neuronal cells, express the receptor structure. The receptor structure is discussed in terms of its role in mediating viral tropism and as an essential cell-surface protein.

Animals↗