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Biomedical subjects

B N Fields

Publications and source records attributed to B N Fields.

At least 91 records · Page 5Linked to original sources
PubMed1985

Identification of a new polypeptide coded by reovirus gene S1.

The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).

Genes, Viral↗
PubMed1985

Genetic reassortment of mammalian reoviruses in mice.

Reassortments between type 1 (Lang) and type 3 (Dearing) reoviruses were isolated from suckling mice infected perorally with an inoculum containing both type 1 and type 3 viruses. A total of five distinct reassortants (designated as E1 through E5) were isolated from animals during the course of the experiment. Two reassortants (E1 and E2) represented the majority of the reassortants isolated. The majority of genes of types E1 and E2 were derived from type 1 (Lang). However, E1 had an M2 gene and an S1 gene derived from type 3 (Dearing), while E2 had M2 and S2 genes derived from type 3 (Dearing). Thus, nonrandom reassortment between mammalian reoviruses can be demonstrated in vivo.

Animals↗
PubMed1985

Differential effects of viral infection on islet and pituitary cell lines.

Although reovirus infection may lead to changes in endocrine function in vivo, little is known about the precise interaction of reovirus with endocrine cells. In this study we have examined the effects of reovirus infection on two types of endocrine cells, GH4C1 cells and RINm5F cells. Both type 1 reovirus and type 3 reovirus infect the two cells lines and appear to grow equally well. Viral replication occurred within the first 24 h following infection after which viral titers remained stable for 3 days. By 48-72 h after viral infection, substantial cytopathic effects were noted in RINm5F cells infected with both type 1 and type 3 reovirus. In GH4C1 cells, type 3 reovirus was most effective in producing cell death, and type 1 reovirus was significantly less cytotoxic despite a similar viral titer. Only type 1 reovirus caused a specific inhibition of overall protein and DNA synthesis, and this occurred only in the RINm5F cells. Over the time course studied, GH4C1 cells successfully infected with type 1 reovirus demonstrated no cytopathic effects, and only minimal alterations in cellular function were noted. Intracellular insulin content and insulin secretion, a "luxury function" of the RINm5F cells, were also surprisingly well maintained in the first 48 h after viral infection. In addition, virally infected cells were able to respond to glyceraldehyde, an insulin secretagogue, although the response appeared to be somewhat blunted compared to that of control cells. These results suggest that viral infection of endocrine cells results in specific alterations that depend on the nature of the infecting virus. In addition, the cellular environment of the host cell may be an important determinant in the outcome of viral infection.

Animals↗
PubMed1985

Cell receptors for the mammalian reovirus. IV. Reovirus-specific cytolytic T cell lines that have idiotypic receptors recognize anti-idiotypic B cell hybridomas.

Cytotoxic T lymphocyte (Tc) cell lines specific for reovirus type 3 lysed an uninfected B cell hybridoma line, 87.92.6, that expresses and secretes an anti-idiotypic antibody that reacts with an anti-viral hemagglutinin monoclonal antibody, 9BG5. Monoclonal anti-idiotype 87.92.6 was shown by fluorescence analysis to specifically bind to reovirus Tc and to block reovirus-specific Tc from killing reovirus-infected target cells or the 87.92.6 hybridoma. An anti-LFA-1 monoclonal antibody, M17, interfered with Tc-mediated lysis of reovirus-infected targets and the 87.92.6 cells, indicating the similarity of cellular interactions mediated by LFA-1 structures when Tc bind to virally infected targets or 87.92.6 targets. Together with studies in which anti-H2 or monoclonal idiotypic antibodies were found to interfere with reovirus-specific Tc recognition of virally infected or 87.92.6 targets, these experiments indicate that some reovirus-specific Tc have conformations in their receptor that can be recognized by anti-idiotype.

Animals↗
PubMed1984

Syngeneic monoclonal antiidiotype can induce cellular immunity to reovirus.

A syngeneic monoclonal antiidiotypic antibody was generated in BALB/c mice after repeated immunization with a BALB/c monoclonal anti-reovirus hemagglutinin (HA) antibody. The resultant syngeneic monoclonal antiidiotypic antibody, in the absence of adjuvant, was found to be capable of priming both BALB/c (H-2d, Igh-1a) and C3H/Hej (H-2k, Igh-1j) mice for Lyt-1+- and Lyt-2+-dependent responses against the mammalian reovirus. By the use of intertypic reassortants and variant virus analysis, the specificity of the response was finely mapped to the neutralization domain of the viral hemagglutinin (HA). Using purified monoclonal antiidiotype, we were able to compare the potency of antiidiotype to virus in terms of induction of immunity. 8 X 10(8) protein molecules were able to prime for cellular responses to reovirus. These studies indicate that in the reovirus system, T cells and B cells share idiotypic configurations, and that antiidiotypic antibodies of the type described herein may be useful in the development of vaccines against certain viral infections.

Animals↗
PubMed1984

Genetic diversity in natural populations of mammalian reoviruses: tryptic peptide analysis of outer capsid polypeptides of murine, bovine, and human type 1 and 3 reovirus strains.

We have studied the structural relationships between the outer capsid polypeptides of eight murine, bovine, and human isolates of type 1 and 3 mammalian reoviruses. Our results show that the outer capsid polypeptides of reoviruses isolated from different mammalian species, in different years and different geographical areas, have both conserved and unique methionine-containing tryptic peptides. We found that tryptic peptides from mu 1C polypeptides of two human, one murine, and two bovine type 3 isolates and one human and two bovine type 1 reoviruses are highly conserved. Our data show that only one tryptic peptide pattern of the mu 1C polypeptide (encoded by the M2 gene) was present in reoviruses isolated from the three different mammalian species. The mu 1C polypeptide of the type 3 Dearing strain contained one tryptic peptide not found in any other reovirus isolate examined. In marked contrast to the mu 1C polypeptides, the sigma 3 polypeptides (encoded by the S4 gene) of three type 1 and three type 3 isolates were divided into two patterns based on significant differences in their tryptic peptides. In addition, at least seven tryptic peptides were conserved among the sigma 3 polypeptides of all virus strains examined. The sigma 3 polypeptide of the type 3 Dearing strain was distinguishable from the sigma 3 polypeptides of all other strains examined. The one mu 1C and two sigma 3 tryptic peptide patterns were found to occur interchangeably in isolates of type 1 or type 3. About 1/3 of the tyrosine-containing tryptic peptides of sigma 1 polypeptides of four type 3 isolates examined were conserved. Comparison of peptide differences in sigma 1 polypeptides of these isolates showed that each had one or more unique tryptic peptides, suggesting that the S1 genes coding for these polypeptides had undergone genetic drift or, alternatively, that there are at least two tryptic peptide patterns present among the sigma 1 polypeptides of these isolates. Our results suggest that genetic drift and reassortment are the most likely explanation for the extensive genetic diversity found in natural populations of mammalian reoviruses.

Animals↗
PubMed1983

Hemagglutinin variants of reovirus type 3 have altered central nervous system tropism.

Variants of the Dearing strain of reovirus type 3 with antigenically altered hemagglutinin proteins are much less neurovirulent than the parental virus. When injected intracerebrally into mice these variants infected a subset of the brain neurons that were infected by the parental virus. When injected intraperitoneally, the variants did not spread to the brain. These results indicate that minor modifications of the reovirus hemagglutinin dramatically alter the ability of the virus to spread into and injure the central nervous system.

Animals↗
PubMed1983

The sigma 1 protein determines the extent of spread of reovirus from the gastrointestinal tract of mice.

After intragastric inoculation of adult mice, type 1 reovirus was initially concentrated in Peyer's patches over the first 4 hr after inoculation, then spread sequentially to the mesenteric lymph nodes and spleen. For type 3 reovirus, however, initial entry into Peyer's patches in adult mice was followed by loss of viral infectivity so that by 4 hr after inoculation virtually no infectious virus was detected in the intestine, and spread to extraintestinal tissues did not occur. In 10-day-old mice, type 3 was capable of spread to the mesenteric lymph nodes but not the spleen. Thus, as animals aged there was a greater restriction of the spread of type 3 from the intestine. Studies using a field isolate of type 3 reovirus that is resistant to intestinal proteases, and genetic studies utilizing type 1 x type 3 viral reassortants, revealed that the viral sigma 1 protein determined the capacity of reovirus to spread from the intestine in both adult and 10-day-old mice. Thus, the interaction of reovirus with host defense mechanisms, and the age-dependent restriction of spread of type 3 reovirus from the intestine are mediated by the viral sigma 1 protein.

Aging↗
PubMed1983

Cytochalasin B inhibits the maturation of measles virus.

The release of measles virus was studied in the presence of cytochalasin B (CB), a drug that disrupts actin microfilaments. In the presence of CB, infected cells accumulated infectious virus while virus released from these cultures decreased drastically (up to 99% inhibition). Electron micrographs showed that viral buds were reduced and had an unusual distribution along the cell membrane in CB-treated cultures. CB inhibition of released virus occurred rapidly (within 30 min) and to a full extent even when the drug was added during the final 2 hr of a 48-hr replicative cycle. CB inhibition of cellular functions is reversible and, similarly, inhibition of virus release could be almost completely reversed within 30 min after the drug was removed. Since CB can also inhibit sugar transport and protein glycosylation, 2-deoxy-D-glucose (DG) was used to study the manifestations of glycosylation inhibition. DG inhibited virus production only when added during the first one-third of the replicative cycle and inhibited cell-associated and released virus to an equal extent. Cytochalasin D, which disrupts microfilaments without affecting protein glycosylation, caused an inhibition of virus release analogous to the inhibition caused by CB. Thus, alteration of microfilament structure alters the normal budding process of measles virions. This suggests that microfilaments may play a role in the release of budding virions.

Animals↗
PubMed1983

A genetic map of reovirus: assignment of the newly defined mutant groups H, I, and J to genome segments.

Mutants representing three previously undefined reovirus type 3 mutant groups have been isolated following backcross of suppressed pseudorevertants to wild type (R.F. Ramig and B.N. Fields, 1979, Virology 92, 155-167; R. Ahmed, P.R. Chakraborty, A.F. Graham, R.F. Ramig, and B.N. Fields, 1980, J. Virol. 34, 383-389). The prototype mutant of each of the three new mutant groups was mapped by analysis of genome segment segregation in intertypic recombinants derived from crosses between the type 3 ts mutants and ts mutants of type 1 or type 2. Segregation analysis revealed the location of the group H prototype mutant tsH(26/8) to be genome segment M1, that of the group I prototype mutant tsI(138) to be segment L3, and that of the group J prototype mutant tsJ(128) to be segment S1. Mapping of the group I and J lesions required the identification of suppressed ts lesions in some of the intertypic rcombinant clones.

Animals↗
PubMed1983

Topological analysis of the reovirus type 3 hemagglutinin.

We previously showed that the reovirus type 3 hemagglutinin (HA) has distinct functional domains. For example, we identified one group of anti-HA monoclonal antibodies which only inhibited virus-mediated hemagglutination and another group which exclusively neutralized reovirus infectivity. Using competition radioimmunoassays, we now report that these functionally discrete domains on the reovirus type 3 HA correspond to discrete antigenic regions of the protein.

Antibodies, Monoclonal↗
PubMed1983

Genetic variation during persistent reovirus infection: isolation of cold-sensitive and temperature-sensitive mutants from persistently infected L cells.

We have examined the evolution of reovirus in two independently established persistently infected (p.i.) cell lines. We found that reovirus undergoes extensive mutation during persistent infection in L cells. However, there was no consistent pattern of virus evolution; in one p.i. cell line temperature-sensitive (ts) mutants were selected, whereas cold-sensitive (cs) mutants were isolated from the second p.i. culture. Neither the cs nor the ts mutants isolated from the carrier cultures expressed their defect at 37 degrees, the temperature at which the p.i. cells were maintained, indicating that the cs and ts phenotypes were nonselected markers. These results emphasize the point that emergence of the ts or cs mutants during persistent infection only signifies that the virus has changed; it does not necessarily imply that the particular mutant is essential for the maintenance of the persistent infection. Given the high mutation rate of viruses, and the wide spectrum of viral mutants present in carrier cultures, it is essential to distinguish the relevant changes from those which may simply represent an epiphenomenon. In the accompanying paper (R. S. Kauffman, R. Ahmed, and B. N. Fields Virology, 130, 79-87, 1983), we show that by using a genetic approach, it is possible to identify the viral gene(s) which are critical for the maintenance of persistent reovirus infection.

Animals↗
PubMed1983

Selection of a mutant S1 gene during reovirus persistent infection of L cells: role in maintenance of the persistent state.

LR-7 cells, variant L cells derived from a type 3 reovirus persistently infected (p.i.) carrier culture (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25, 325-332, 1983) were used to define the viral genes critical for maintenance of the persistent state. A cloned viral isolate (L/C virus) derived from the p.i. culture replicated normally in LR-7 cells, while wild-type (wt) viruses of the three reovirus serotypes replicated less efficiently. To identify the viral gene(s) permitting enhanced replication of L/C virus in LR-7 cells, viral reassortants were prepared by mixed infection of L cells with L/C virus and type 1 wt. Study of the one-step growth curves and final yields of large numbers of reassortants in both L cells and LR-7 cells revealed that the presence of the S1 gene from L/C virus was critical for normal viral replication in LR-7 cells. However, this phenotype was suppressed by the simultaneous presence in reassortants of both the M2 and S4 genes from the type 1 wt parent. The critical change in the S1 gene occurred by passage 13 (63 days) after initiation of the carrier culture. Although multiple mutations are present in the viral population from p.i. cultures, certain specific mutations can be identified as critical for maintenance of the persistent state.

Animals↗
PubMed1983

Interactions of preimplantation mouse embryos with reovirus serotypes 1 and 3.

Mouse two-cell embryos were infected in vitro with reovirus serotypes 1/Lang and 3/Dearing, and the embryos were either implanted into pseudopregnant mice or observed in vitro for cytopathic effects. The reovirus serotypes 1/Lang and 3/Dearing differed in their capacity to kill embryos in vitro and in vivo: when embryos were infected in vitro with reovirus serotype 1/Lang and then transferred to foster mothers, pups resulted only at multiplicities of infection of a few particles per embryo. In contrast, infection of embryos with as much as 6 X 10(4) reovirus type 3 particles per embryo resulted in viable pups. In vitro, reovirus serotype 1/Lang was more virulent than serotype 3/Dearing. The infection of ovum with reovirus offers a unique model for the study of congenital infection and should yield important information concerning the molecular basis of virus virulence to maturing fetuses.

Animals↗
PubMed1983

Specific plasma membrane receptors for reovirus on rat pituitary cells in culture.

Specific cellular and host tropism is a characteristic property of many viruses mediated by the interaction of viral attachment proteins with components of the plasma membrane of the cell. We have studied the binding of virus to cells quantitatively by using type 3 reovirus labeled with 125I and GH4C1 pituitary cells in culture. Binding was rapid at both 4 degrees and 15 degrees C and was stable over a 9-h period. Unlabeled virus inhibited binding of the labeled virus in a dose-dependent manner. Scatchard analysis revealed 4,200 viral binding sites/cell with an apparent affinity of 1.2 X 10(-11) M. Also, binding of type 3 reovirus was inhibited by antibodies directed against the viral hemagglutinin and partially inhibited by type 2 reovirus, but was unaffected by type 1 reovirus or a variety of other ligands that bind to receptors on GH4C1 cells. These data indicate that reovirus binds to a high affinity, specific receptor on target cells, which may control its tropism and ultimate disease expression.

Animals↗