Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Professor L.A. Prevec


Whereas the Vesiculovirus genus of the Rhabdoviridae is comprised of several more or less biochemically distinguishable serotypes, a study was undertaken to assess conservation of protein structure in Vesiculovirus members. It was of particular interest to assess protein structural conservation for Piry and Chandipura viruses which show only slight serological cross-reactivity with VS Indiana, the prototype of the genus. Three methods of peptide mapping were employed: tryptic peptide mapping by 2-dimensional thin-layer electrophoresis-chromatography, a novel mapping procedure which utilized limited cleavage of tryptophan peptide bonds with the chemical agent N-chlorosuccinimide (NCS) followed by fractionation using SDS-polyacrylamide gel electrophoresis, as well as an adjunct to the second method where proteins labeled at their amino-termini with f[³⁵S]Met were used to derive linear oriented maps of tryptophan positions. The methionine-containing tryptic peptides of the 5 constituent proteins of members of the Vesiculovirus: VS Cocal, VS New Jersey (Missouri and Concan strains), and Piry viruses were compared to the prototype member, VS Indiana (T) standard strain, by two-dimensional thin-layer chromatography-electrophoresis. While the corresponding proteins of each virus isolate could be identified by their characteristics peptide map there were common peptides shared among the members tested. On comparison with the prototype VS Indiana(T), VS Cocal possessed common peptides from its N, M, and G proteins whereas VS New Jersey possessed common peptides from N and M proteins. Pity virus possessed one N protein peptide in common with the prototype. Comparison of the tryptic peptides of 2 members of the VS New Jersey serotype but from separate subtypes revealed characteristic differences between these closely related viruses. The partial cleavage products resulting from NCS treatment of [³⁵S] methionine-labeled Vesiculovirus proteins were analyzed. The resulting cleavage patterns of the N and M proteins of VS Indiana(T), VS Cocal, VS New Jersey Missouri and Concan strains, Chandipura and Piry, while unique, showed a number of similarities in peptide fragment position and pattern. Pattern similarity was detectable among all the NCS patterns of the N and M proteins, but less so in the case of the Chandipura M protein. The most pronounced similarity was observed among the N protein NCS patterns of the VS viruses or between Piry and Chandipura. The M protein NCS patterns did not demonstrate as unified a relationship among Vesiculovirus members as the N protein NCS patterns, but the same two groupings of members could be made.

Cleavage of f[³⁵S]Met-labeled N and M proteins of Vesiculovirus members with NCS and subsequent SDS-PAGE separation allowed the derivation of linear oriented maps of tryptophan positions. Visual comparison of linear peptide maps showed extensive conservation of tryptophan residues among N and M proteins of the VS viruses Indiana(T), Cocal, and New Jersey (M) as well as Piry virus. The VS New Jersey (M) and Pity tryptophan map of N protein were very homologous, both containing 6 sites in similar positions. There 6 tryptophan residues appeared to be conserved in VS Indiana(T) and VS Cocal, although these two viruses contained additional tryptophan sites. The M proteins of VS Indiana(T), VS Cocal, VS New Jersey(M) and Piry possessed 3 common tryptophan positions, although there seemed to be some form of molecular rearrangement in the case of Piry virus.

Considered in isolation, the tryptic peptide mapping analysis supports the existing serological interrelationships by demonstrating VS Indiana(T) to be closely related to VS Cocal and less related to VS New Jersey but equivocally related to Piry virus. By employing the NCS peptide mapping technique, paritcualrly when used to prepare linear oriented maps, an ancestral relationship was demonstrated among all of the Vesiculoviruses tested. However, Chandipura and Piry viruses could be distinguished from the VS viruses suggesting a closer ancestral relationship among members within rather than between these groups.

NCS peptide mapping proved to be a valuable method of structural comparison which complemented tryptic peptide analysis under the conditions employed. Tryptic peptide mapping was much more discriminatory than NCS peptide mapping and as a consequence the former was ideal for comparison of closely related members whereas the latter method was a powerful means of detecting homology among more distantly related organisms. Given the fact that genus classification within the Rhabdoviridae is primarily serologic, relying on conservation of protein structure, this study supports the classification of the International Committee on Taxonomy of Viruses since it confirms the existence of conserved protein structures among members of the Vesiculovirus.

The D₁N protein abnormality is manifest by an increase in electrophoretic mobility in SDS polyacrylamide gels, suggesting a decrease in apparent molecular weight of 1,000. Some of the previously described methods were employed for the elucidation of the structural basis of the N protein abnormality in the VS New Jersey(M) D₁ temperature-sensitive mutant. It was hypothesized that the D₁ protein defect resulted from premature termination due to nonsense mutation. The N protein alteration was located on the linear map of methionine and tryptophan residues as determined by NCS and CNBr cleavage of protein labeled at its amino-terminus. This approach allowed identification of a region of ca. 2-3K molecular weight at the C-terminal end of the molecule which was required to detect the alteration. This was confirmed by removal of a similar sized portion of the C-terminal region with carboxypeptidases A and B. The tryptic peptides from the C-terminal region were identified by carboxypeptidase A plus B sensitivity and the C-terminal amino acid and tryptic peptide were identified after carboxypeptidase B treatment. The C-terminal amino acid of mutant, revertant and wild type was identified as lysine and the C-terminal tryptic peptide was indistinguishable among them. The carboxypeptidase A plus B susceptible tryptic peptides were indistinguishable as well, but a carboxypeptidase A plus B resistant tryptic peptide was altered in the D₁ N protein and was restored to wild type on reversion. Mutant N protein also differed from wild type in isoelectric point, but reversion resulted in restoration of wild type isoelectric point. From these data it was concluded that the D₁ N protein alteration was not a premature termination, but was probably a missense mutation which results in an apparent decrease in molecular weight as detected on SDS-PAGE. It was concluded that the protein defect was not contained in the C-terminal region (ca. 2K MW), but that this portion of the molecule was required to detect the abnormality on SDS-PAGE. The region of the molecule containing the alteration was located on the linear physical map and models are presented to explain the increased mobility on SDS-polyacrylamide gel electrophoresis.

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