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M J Buchmeier

Publications and source records attributed to M J Buchmeier.

At least 73 records · Page 4Linked to original sources
PubMed1987

Analysis of the genomic L RNA segment from lymphocytic choriomeningitis virus.

The arenavirus genomic L RNA segment represents approximately 70% of the viral genetic material but current understanding of the organization, regulation, and functioning of the viral L products remains limited. Biological studies with reassortant viruses have implicated the L RNA segment in the lethal infection of adult guinea pigs produced by LCMV-WE but no further explanation of the pathogenic process is presently available. We have initiated a detailed molecular analysis of LCMV L products based on construction and characterization of L-specific cDNA clones and synthesis of L-specific hybridization probes. Nucleotide sequencing studies have allowed the derivation of a partial amino acid sequence for a predicted L protein and antisera raised against synthetic peptides have demonstrated an L protein in Western blotting experiments. Using this approach we have identified a single high molecular weight protein (approximately 200,000 Da) in purified virions and in viral ribonucleoprotein complexes extracted from acutely infected tissue culture cells. This L protein is translated from a nonpolyadenylated, genomic complementary L mRNA and potentially represents part or all of the viral RNA-dependent RNA polymerase.

Amino Acid Sequence↗
PubMed1987

Molecular characterization of the genomic S RNA segment from lymphocytic choriomeningitis virus.

We have used cDNA clones derived from the genomic S RNA segment of lymphocytic choriomeningitis virus (LCMV), Armstrong strain, as hybridization probes to monitor virus gene expression during acute infections. Our results with strand-specific probes confirm the ambisense character of the LCMV S RNA segment and document the presence of both genomic sense and genomic complementary sense RNA species over the time course of infection. We have used nucleotide sequence information to predict primary amino acid sequences for the major viral structural proteins, nucleoprotein (NP) and glycoprotein (GP-C). Antibodies raised against synthetic peptides derived from these predicted protein sequences have indicated that the gene order for the S segment is 3' NP----5' GP-C and provided direct demonstration that the GP-1 portion of the GP-C precursor is encoded nearest the 5' end of the S segment. Comparison of the predicted amino acid sequences for NP and GP-C between the Armstrong CA-1371 strain and the WE strain shows over 90% amino acid identity. This suggests that significant differences described for the pathogenic potential of the Arm and WE strains in C3H mice reside in one or a very few critical amino acid changes.

Amino Acid Sequence↗
PubMed1987

Site-specific antibodies define a cleavage site conserved among arenavirus GP-C glycoproteins.

Arenaviruses share a common strategy for glycoprotein synthesis and processing in which a mannose-rich precursor glycoprotein, termed GP-C in lymphocytic choriomeningitis virus (LCMV), is posttranslationally processed by oligosaccharide trimming and proteolytic cleavage to yield two structural glycoproteins, GP-1 and GP-2. Mapping the orientation and proteolytic cleavage site(s) in such polyproteins has traditionally required direct protein sequencing of one or more of the cleaved products. This technique requires rigorous purification of the products for sequencing and may be complicated by amino-terminal modifications which interfere with sequence analysis. We used an alternative approach in which synthetic peptides corresponding to sequences bracketing a potential protease cleavage site were used to raise antisera which define the boundaries of the cleaved products. We found that cleavage of LCMV GP-C to yield GP-1 and GP-2 occurs within a 9-amino-acid stretch of GP-C which contains a paired basic amino acid group -Arg-Arg-, corresponding to amino acids 262 to 263 in the LCMV GP-C sequence. By comparison with the predicted amino acid sequences of a second LCMV strain, LCMV-WE, as well as with the deduced amino acid sequences of the New World arenavirus Pichinde and the Old World virus Lassa, we observed similar conservation of paired basic and flanking amino acid sequences among these viruses.

Amino Acid Sequence↗
PubMed1987

Immuno-gold localization of prion filaments in scrapie-infected hamster brains.

The brains of scrapie-infected hamsters have been examined for the presence of structures antigenically related to the prion protein (PrP 27-30). Glutaraldehyde-perfused hamster brains, 72 days postinfection, were immunostained using rabbit monospecific antisera raised against synthetic peptides corresponding to the N-terminal 13 or 15 amino acids of PrP 27-30, and using rabbit antisera raised against infectious prions or PrP 27-30 purified from scrapie-infected hamster brains. Antisera to the synthetic peptides stained extracellular filaments in agreement with previous immunoperoxidase studies which used affinity-purified PrP 27-30 antibodies; in addition to subependymal and subpial localization, we show ventricular and perivascular staining. Using a colloidal gold-secondary antibody technique, we have demonstrated that the antibodies labeled filaments measuring 7 to 17 nm in diameter. Whereas most of the periventricular and perivascular filaments appeared extracellular, some appeared to be within processes intimately associated with ependymal cells, degenerating membranes of astrocytes, and neurites.

Amino Acid Sequence↗
PubMed1987

Catabolism of homologous murine monoclonal hybridoma IgG antibodies in mice.

Two neutralizing monoclonal hybridoma antibodies to the surface spike glycoprotein E2 of the JHM strain of murine hepatitis virus were injected into homologous BALB/c mice, and their biological half-lives were determined by sequential titration of plasma samples in a virus-specific enzyme immunoassay. Intravascular half-lives of monomeric immunoglobulins were estimated at 8.0 +/- 1.5 days for antibody 5B19.3, an IgG1, and 12.7 +/- 2.4 days for antibody 4B11.6, an IgG2a. These catabolic rates are statistically different from each other (P less than 0.001) and significantly higher than previously reported values, which were all obtained with radiolabelled polyclonal or myeloma immunoglobulins of unknown specificities. Failure to remove aggregated 4B11.6 antibodies by high-speed centrifugation yielded a statistically significant acceleration of biological turnover (half-life 9.9 +/- 1.6 days; P less than 0.01).

Animals↗
PubMed1986

Dissecting the molecular anatomy of the nervous system: analysis of RNA and protein expression in whole body sections of laboratory animals.

Nucleic acid hybridization and protein blotting procedures have allowed the specific detection of both RNA and protein gene products in whole body sections of a selected host. These procedures permit efficient and reproducible screening of both endogenous and exogenous (viral) gene products, thus facilitating the study of normal differentiation, the localization of virus and the monitoring of viral diseases. Although success with RNA hybridization has thus far been limited to exogenous viral gene expression, the protein immunoblotting procedures have the sensitivity to detect endogenous protein products with high resolution. These combined procedures should prove useful for the study of protein expression in numerous developmental systems.

Animals↗
PubMed1986

Proteins of lymphocytic choriomeningitis virus: antigenic topography of the viral glycoproteins.

Topographical relationships among antigenic sites on the envelope glycoproteins of lymphocytic choriomeningitis virus (LCMV) were established using a panel of monoclonal antibodies (MAb) directed against viral GP-1 and GP-2. Purified MAb were radioiodinated and used as probes in a solid phase competitive binding assay. Epitopes on LCMV GP-1 were found to cluster in four antigenic sites. Five neutralizing MAb raised by immunization with the WE strain of LCMV reacted with a single topographic site, termed GP-1A, which was present on four strains of LCMV examined in this study. A second site, GP-1B, was characterized by two MAb which partially competed with one another and with a subset of neutralizing antibodies. This site appeared to be close to site A and was found to be nonneutralizing. The third site, GP-1C, contained sequential epitopes and was also nonneutralizing. Antibodies binding to site B enhanced the binding of MAb at site C, presumably through a conformational change. In addition to the common neutralizing site A, LCMV Armstrong strain (LCMV-Arm) GP-1 contained a second topographically related neutralizing site, GP-1D, which was specific for LCMV-Arm, absent in WE, and appeared to be the major immunogenic epitope on GP-1 of this virus. Analysis of MAb binding to LCMV GP-2 demonstrated the presence of three overlapping binding sites. GP-2A was defined by two antibodies while GP-2B and C represented binding sites of one antibody each. Guinea pigs primed with LCMV-Arm and challenged with LCMV-WE developed a significant immune response which was directed toward the common major neutralizing site, GP-1A, but had poor responses to the LCMV-Arm specific neutralizing site GP-1D. Immune sera contained antibody to site GP-1B but lacked detectable antibody to GP-1C.

Animals↗
PubMed1986

Molecular mimicry: frequency of reactivity of monoclonal antiviral antibodies with normal tissues.

More than 600 monoclonal antiviral antibodies made against 11 different viruses were screened against 14 different organs from normal uninfected mice. Of these antiviral antibodies, 21, or approximately 3.5%, reacted with specific cells in these organs. Several of these antibodies were of the multiple-organ-reactive type and recognized antigens in more than one organ. It was concluded that the reactivity of monoclonal antiviral antibodies with normal tissues is a common phenomenon.

Animals↗
PubMed1986

Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence.

Strains of the murine coronavirus mouse hepatitis virus type 4 (MHV-4) which contained a mutation in the E2 peplomer glycoprotein were obtained by selection for resistance to neutralization by monoclonal antibodies. Characterization of six variants representing two independent epitopes on E2, E2B and E2C, by in vitro neutralization and antibody-binding assays demonstrated that selection for an alteration in epitope E2B also resulted in changes in epitope E2C and vice versa. We observed a mutation frequency of approximately 10(-4.3) to 10(-4.6), which is consistent with the expected occurrence of single point mutations. The variant virus strains were attenuated with respect to neurovirulence when compared with wild-type MHV-4. Mice normally develop encephalomyelitis and die after wild-type MHV-4 infection. Mice receiving 2- to 3-log-higher doses of the variant strains survived and developed demyelinating disease. As the disease progressed, evidence of remyelination and ongoing demyelination was observed up to 65 days after infection. Virus reisolated 15 days after infection retained the variant phenotype. The data indicate that the E2 glycoprotein plays a central role in determining the cellular tropism and virulence of MHV-4 in the mouse.

Animals↗
PubMed1986

Experimental models of virus attenuation and demyelinating disease.

Several lines of evidence suggest that viruses may be aetiologically related to human demyelinating disorders, but no clear causal relationships have been established. In an effort to understand the pathogenesis of virus-induced CNS disease, animal model systems have been employed. Recent results indicate that mutational changes in viral surface components may profoundly affect the course and outcome of disease.

Animals↗
PubMed1985

Perturbation of differentiated functions during viral infection in vivo. I. Relationship of lymphocytic choriomeningitis virus and host strains to growth hormone deficiency.

Disordered growth and glucose metabolism secondary to growth hormone deficiency is associated with persistent lymphocytic choriomeningitis virus (LCMV) infection. C3H/St, BALB/WEHI, and SWR/J mice infected at birth with LCMV:ARM carried virus in their blood and organs throughout life but only C3H/St mice developed growth hormone insufficiency. BALB/WEHI and SWR/J infected mice contained normal amounts of growth hormone in their pituitaries and a relatively small proportion of the cells containing growth hormone replicated the virus. In susceptible C3H/St mice, the disease-causing viral strains (LCMV:ARM, E-350, and Pasteur) replicated to higher titers and infected the vast majority of cells producing growth hormone in the anterior lobe of the pituitary. In contrast, LCMV strains Traub and WE replicated in far fewer growth hormone-producing cells and failed to disorder growth hormone synthesis. In another paper (Y. Riviere, R. Ahmed, P. Southern, and M. B. A. Oldstone (1985), Virology 142, 175-182) these findings are used to make reassortants between LCMV:ARM (disease positive) and LCMV:WE (disease nil) and the pathogenic effect is mapped to the small RNA segment of LCMV:ARM. Peptides cleaved by trypsin and chymotrypsin from growth hormone molecules isolated from infected cells or control cells were equivalent when examined by two-dimensional electrophoresis. Further, transfer of antibody to interferon failed to alter the growth hormone insufficiency in these mice, although it corrected LCMV-induced liver disease of BALB mice, suggesting that interferon did not play a dominant role in this disease. The selective tropism of LCMV:ARM for cells containing growth hormone over cells that contain prolactin was observed in both infected animals and in cultured GH-3 cells.

Animals↗
PubMed1985

Antigenic variation among murine coronaviruses: evidence for polymorphism on the peplomer glycoprotein, E2.

A panel of 28 monoclonal antibodies (MAb) against the structural proteins of murine hepatitis virus-4, strain JHM (MHV-4) was used in three antigen binding assays to determine the extent of antigenic homology among six strains of murine coronaviruses. The antigenic determinants studied were highly conserved on the E1 glycoproteins and nucleocapsid (N) proteins of all strains tested. In contrast, antigenic polymorphism was observed among the E2 glycoproteins. Of three previously described antigenic determinants against which neutralizing antibodies are directed, only one, termed A(E2), was conserved on all strains. Antigenic site B(E2) was found only on the strongly neurotropic MHV-4 and site C(E2) was present on the virulent MHV-4 and MHV-3 (hepatotropic) strains, but absent on the weakly pathogenic MHV-A59, MHV-1 and MHV-S strains. Four non-neutralizing antibodies against at least one topographically distinct antigenic determinant, which we previously designated D(E2), gave binding patterns consistent with two distinct sites. One of these was present on all MHV strains tested and the other was present on all strains except MHV-S. These non-neutralizing antigenic sites were redesignated E(E2) and D(E2) respectively.

Antibodies, Monoclonal↗
PubMed1985

Properties and characterization of monoclonal antibodies to Tacaribe virus.

Monoclonal antibodies prepared against Tacaribe and Junin viruses have been used to define further the serological relationships between arenaviruses of the Tacaribe complex. A close relationship was found between these two viruses and the heterologous Amapari and Machupo viruses, with Pichinde virus and Parana virus being more distantly related. Among the antibodies specific for Tacaribe virus, five were found to react with viral antigens at the surface of infected cells and to neutralize virus infectivity in vitro. These five antibodies could be differentiated by competitive immunoassay as recognizing at least two antigenically distinct epitopes. The kinetics of reaction between antibody and virus were examined for all five neutralizing antibodies. One antibody (2.25.4) effectively neutralized all infectious virus. The remaining four directed against a second epitope gave significant persistent fractions which could be reduced by addition of complement, anti-mouse immunoglobulin, or antibody 2.25.4. Variants of Tacaribe virus resistant to neutralization by antibody 2.25.4 were obtained by growth in the presence of this antibody and neutralization kinetics were reexamined using the heterologous monoclonal neutralizing antibodies. Several different neutralization profiles were obtained, suggesting that point mutations resulted in conformational changes at topographically selected distinct epitopes recognized by the remaining antibodies.

Animals↗