Clifford W. Bond

Antibody Imprint of a Membrane Protein Surface PHAGOCYTE FLAVOCYTOCHROME b

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody “imprint” of the Cyt bsurface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed sequences. The NMR data supported the phage-display analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29TAGRF33 and183PQVNPI188 from discontinuous regions of p22. Although these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the transmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cytb surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.

Biological properties of mengovirus: Characterization of avirulent, hemagglutination-defective mutants

SummaryBiological properties of two mengovirus mutants, 205 and 280, were compared to those of wild-type virus. The mutants exhibited alterations in plaque morphology, hemagglutination, and virulence in mice, but were not temperature-sensitive. Agglutination of human erythrocytes by mengovirus was dependent on the presence of sialic acid on the erythrocyte surface; however, free sialic acid failed to inhibit hemagglutination. Glycophorin, the major sialoglycoprotein of human erythrocyte membranes, exhibited receptor specificity for wild-type virus, but not for mutants 205 or 280. Cross-linking studies indicated that glycophorin exhibited binding specificity for the alpha (1 D) structural protein. The LD50 titers for wild-type mengovirus were 7 and 1500 plaque forming units (PFU) in mice infected intracranially (IC) and intraperitoneally (IP), respectively. However, mice infected IC or IP with 106 or 107 PFU of mutant 205 or 280 did not exhibit symptoms indicative of virus infection. Revertants were isolated from the brains of mice infected with mutant 205, but not from the brains of mice infected with mutant 280. The biological characterization of the revertants indicated that hemagglutination and virulence may be phenotypically-linked traits.

A rapid, quantitative assay for titration of bovine virus diarrhoea-mucosal disease virus☆

Abstract An end point dilution microtitration assay is described that can be used for the titration of both cytopathic and non-cytopathic isolates of bovine virus diarrhoea-mucosal disease virus. Indirect immunofluorescence is used to detect infected MDBK cells in the wells of Terasaki plates. The virus titre is derived from the number of uninfected wells, using the Poisson distribution. The assay is simple, fast and economical. Titres of cytopathic virus determined by the microtitration assay and standard plaque assay are equivalent.

Biological and macromolecular properties of murine cells persistently infected with MHV-JHM

SummaryA persistently-infected neuroblastoma culture [Neuro-2A (JHMV)] was established with the murine hepatitis virus JHM [MHV-JHM]. After 100 days of passage, the endogenous virus [Neuro-2A (JHMV) end] released by this culture was unable to induce the syncytia typical of MHV-JHM and the endogenous virus was not temperature-sensitive. The Neuro-2A (JHMV) culture was cured of virus production by passage under neutralizing antibody [Neuro-2A (JHMV) Ab]. The Neuro-2A (JHMV) and the Neuro-2A (JHMV) Ab cultures were as susceptible to heterologous infection with mengovirus and vesicular stomatitis virus as the uninfected Neuro-2A culture. However, the Neuro-2A (JHMV) and Neuro-2A (JHMV) Ab cultures were partially resistant to homologous superinfection by MHV-JHM and the closely related MHV-A59. Virus related to MHV-JHM was rescued from the antibody-cured cells by cell fusion. The synthesis of MHV-JHM specific antigens by Neuro-2A (JHMV) cells, Neuro-2A (JHMV) Ab cells and 17 Cl-1 cells infected by Neuro-2A (JHMV) end was studied by SDS-PAGE. The genomic RNAs of MHV-JHM and Neuro-2A (JHMV) end were compared by oligonucleotide mapping. The results of the protein and RNA studies indicated that the genome of Neuro-2A (JHMV) end was substantially modified from the genome of MHV-JHM, but the modifications did not significantly alter the molecular size of the viral-specific proteins.

Structural and physiological properties of mengovirus: Avirulent, hemagglutination-defective mutants express altered alpha (1 D) proteins and are adsorption-defective

SummaryStructural and physiological properties of two mutants of mengovirus, 205 and 280, were compared to those of wild-type virus to understand the molecular basis of changes exhibited in their biological function. Two dimensional gel electrophoresis of wild-type and mutant structural proteins revealed alterations in the isoelectric character of the alpha (1 D) protein of both mutant 205 and 280. These data suggest that alterations in the alpha (1 D) protein may be responsible for the phenotypic changes by the mutants. A delay in detectable virus-specified protein synthesis was exhibited in mutant-infected cells in comparison to wild-type. The amount of RNA synthesized in mutant- and revertant-infected cells was less than that synthesized in wild-type infected cells. Changes in virus-specified macro-molecular synthesis in mutant and revertant-infected cells reflected a decrease in the ability of the viruses to attach to cells.

Protein synthesis in cells infected by murine hepatitis viruses JHM and A59: Tryptic peptide analysis

SummaryThe structural and intracellular proteins of the murine hepatitis viruses MHV-JHM and MHV-A59 were studied by tryptic peptide mapping. The results demonstrated that the virions contained three distinct proteins: the two related chains of the E2 complex, the nucleocapsid protein and the heterogeneous E1 complex. Five distinct virus-specific proteins were synthesized by infected cells. Three of the five intracellular proteins contained tryptic-peptides with properties similar to the three structural proteins. Models describing the evolution of the proteins are proposed. Although the pathogenic properties of MHV-JHM and MHV-A59 differ greatly, the tryptic peptide maps of the corresponding proteins of these MHV strains were remarkably similar.

Relatedness of Virion and Intracellular Proteins of the Murine Coronaviruses JHM and A59

Coronaviruses are a group of RNA viruses with positive polarity that cause a wide variety of diseases in many species including man. The structural proteins of coronaviruses have been studied extensively (reviewed in Robb and Bond, 1979). However, little attention has been focused on the intracellular proteins (Bond et al. 1979, Anderson et al. 1979, Siddel et al. 1980).