G.W. Wagner

Structures derived from cowpea chlorotic mottle and brome mosaic virus protein

Abstract The behavior at different salt and acidic pH levels of protein isolated from three spherical plant viruses was investigated. Broad bean mottle virus protein did not aggregate into recognizable organized structures. Protein from brome mosaic virus formed capsids similar to those of the virus at a pH and salt-dependent efficiency with best yields at pH 5.0 in 0.2 M NaCl. Best yields for cowpea chlorotic mottle virus protein capsids occurred at pH 5.0 in 0.1 M NaCl or from pH 3.5 to 5.0 in 0.2 M NaCl. Protein from the latter virus also formed double-shelled and rosettelike particles in 0.2 M NaCl from pH 5.3 to 5.7. Narrow tubes were made at pH levels of 6.0 and higher, and the tubes were accompanied by T = 1 and T = 3 particles if ribonu-clease-digested RNA was present. In the absence of NaCl in 0.01 M acetate buffer at pH 4.0 and 4.5, laminar and platelike aggregates were found with CCMV protein. The production and some of the properties of the various forms are described.

Inhibitors of a rat brain enkephalin aminopeptidase.

Abstract: Eight protease inhibitors of microbiological origin were examined as potential inhibitors of a homogeneous rat brain enkephalin aminopeptidase. Bestatin [(2S,3R)‐3‐amino‐2‐hydroxy‐4‐phenylbutanoyl]‐l‐leucine and analogs of bestatin having basic, acidic, and other neutral amino acids substituted for the Leu residue exhibited inhibition constants ranging from 3.3 ± 10−5 to 8.3 ± 10−8m. The best inhibitor had a positively charged amino acid (Lys) substituted for Leu. A series of phenylalanyl dipeptides were examined as substrates with the aminopeptidase. The amino acid residue on the carboxyl side of the peptide bond undergoing cleavage was varied systematically in the dipeptides to include neutral, acidic, and basic residues. Again, a positively charged amino acid (Arg) adjacent to the bond undergoing scission was kinetically preferred. These results may be used to design highly specific inhibitors of the enkephalin aminopeptidase.

The self-assembly of spherical viruses with mixed coat proteins.

Abstract Coat protein subunits from cowpea chlorotic mottle virus, brome mosaic virus, and broad bean mottle virus have been mixed in various combinations and ratios and added to RNA to form spherical particles with mixed protein coats. These particles, some of which are infective and resistant to snake venom phosphodiesterase, are immunoelectrophoretically distinct from nucleoproteins with any single type of coat protein.

Biochemical and biophysical properties of two strains of mosquito iridescent virus.

Abstract Properties of “R” and “T” strains of mosquito iridescent virus (MIV) were investigated. Several differing biophysical properties of the two strains were observed due to size differences. The electrophoretic mobility curves of the two strains were somewhat different although the isoelectric points of pH 3.15 and pH 3.30 for RMIV and TMIV, respectively, were similar. The two strains appeared to be antigenically identical but the per cent protein, DNA, and lipid of the two were dissimilar. Other than size variation, no morphological differences between RMIV and TMIV could be detected.

Proteins of Two Strains of Mosquito Iridescent Virus

A comparison of the proteins of ‘R’ and ‘T’ strains of mosquito iridescent virus (M1V) was made using PAGE and amino acid analysis. The banding patterns of RMIV and TM1V were similar, each showing 9 proteins. None of the proteins reacted with Schiff’s reagent. When the proteins of RMIV and TMIV were S-carboxymethylated, PAGE showed only 4 proteins for both strains compared with the 9 bands detected in control preparations. Amino acid analyses and tryptic peptide analyses indicated that the proteins of the two strains were very similar.

Pathology of mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of Aedes aegypti

Abstract A bioassay of mosquito iridescent virus (MIV) of Aedes taeniorhynchus was developed using cell cultures of Aedes aegypti. The dilution end point technique was based on the occurrence of cytopathic effects which were optimum at 31°C. Pelegs A. aegypti cell line was more sensitive and reliable than Singhs A. aegypti cell line for infectivity titration of the “R” and “T” strains of MIV. The highest tissue culture infectivity dose 50s (TCID50) were elicited by virion:cell ratios of approximately 10. TCID50 titers were significantly reduced by virus neutralization with either homologous or heterologous antiserum to either RMIV or TMIV. The virus propagated in either cell line was not infectious to A. taeniorhynchus larvae, or to the respective cells from which the virus was produced. All plaque assay attempts were unsuccessful.

A comparison of the DNA of the “R” and “T” strains of mosquito iridescent virus

Abstract The buoyant density (1.7135 g/cm 3 ), percentage GC (53.9%), and melting temperature ( T m = 76.4°) of the DNA of “regular” mosquito iridescent virus (RMIV) and of “turquoise” mosquito iridescent virus (TMIV) are similar although the molecular weights of the two DNAs are different; 243.3 × 10 6 and 286.7 × 10 6 , respectively. Analyses show that RMIV contains two identical duplex DNA molecules which are about 15% smaller than the single duplex DNA molecule of TMIV. Reassociation studies show that RMIV and TMIV contain about 17 and 30% repetitious DNA, respectively. Homology studies show the two DNAs to be 100% homologous in their DNA nucleotide sequences. We conclude that the DNA of RMIV and TMIV contain identical sequences and that the portion of the TMIV-DNA molecule accounting for the higher molecular weight is composed of repeated sequences common to both strains.

Production and characterization of the cores of the “R” strain of mosquito iridescent virus

Abstract Cores of “R” strain of mosquito iridescent virus (RMIV) were produced in vitro by reacting intact virus with chymotrypsin. Isolation of the cores from undegraded virus and outer capsid protein was accomplished by density gradient and differential centrifugations. Negatively stained core particles had a diameter of 176.1 ± 6.0 nm when examined in the electron microscope. The density of the particles as measured in cesium chloride gradients was 1.33, and the s 20,w was 3126 as measured in the analytical centrifuge. The molecular weight of the cores was calculated to be 1.84 × 10 9 daltons. Protein, DNA, and lipid analysis of the cores accounted for all but 48.0% of the protein of intact virus particles. SDS-polyacrylamide gel electrophoresis of the cores compared with that of intact virus showed that only a 55,000 molecular-weight protein was absent in the former. The cores did not infect larvae or an Aedes aegypti cell line, and serological comparisons of intact virus and cores showed the two did not share common surface antigens.

Characterization of the top component of the “T” strain of mosquito iridescent virus☆

Abstract The molecular weight of the top component of TMIV was found to be 1.94 × 10 9 . The particles contain between 5.0 and 5.7% DNA, 4.3% lipid, and 87.1% protein. The antigenicity and electrophoretic mobility of top component differ from those of TMIV, although the number and molecular weight of top component proteins as determined in polyacrylamide gels appear identical to those of the virus. Top component particles are neither infectious nor do they affect the infectivity of virus suspensions.