The long term goals of the experiments outlined in this proposal are to investigate the proteolytic processing of vaccinia virus (VV) core proteins with regard to: (i) identification of the endo- and/or exoproteolytic pathways required for the maturation of VV core proteins; (ii) analysis of the precursor protein determinants which specify the correct site(s) of proteolytic processing; (iii) identification and molecular examination of the protease(s) responsible for these reactions; (iv) elucidation of whether, and how, the substrate-protease interaction is regulated in vivo; and, (v) determination of the role processed VV core proteins play in assembly of progeny viral particles. Three of the major VV core constituents proteins 4a, 4b, and 25K are late viral proteins produced as precursor polypeptides, p4a, p4b and p25K which are subjected to proteolytic cleavage during viral morphogenesis. Since previous work in our has indicated this proteolysis step plays a very central and important role in the assembly of infectious virions, we now propose to directly analyze this reaction. The map location of these loci and their nucleotide sequences have recently been determined. This information will be used to design fusion protein and synthetic peptide antigens with which to generate epitope-specific antisera for use in concert with immuoprecipitation, western blotting, and immunofluorescence procedures to establish the sequence, kinetics, and cytolocalization of the processing reactions which occur within the infected cell. Furthermore, comparison of amino acid sequences predicted from the derived nucleotide sequences of p4a, p4b, and p25K with the N-terminal amino acid sequences of mature 4a, 4b, and 25K proteins (determined by microsequencing procedures) should identify the portions of the precursors which contain potential cleavage sites. This information in concert with site-directed mutagenesis and marker transfer techniques will be used to dissect the important cis recognition features of these cleavage sites. Similarly, plasmid expression vectors will be utilized to preparatively produce precursor proteins for use in in vitro and in vivo cis and trans processing assays designed to identify the proteinase(s) which catalyze these reactions. If, as is likely, these functions are virus-encoded, the genes will be mapped and subjected to detailed molecular genetic analyses. It is likely that the results of these experiments will provide considerable insight regarding proteolytic processing during VV replication. Given the ability to readily carry out directed genetics on VV-encoded gene products, this information should enable detailed structure-function studies on the molecular mechanisms of proteolysis to be undertaken.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI029367-04
Application #
3144149
Study Section
Virology Study Section (VR)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Oregon State University
Department
Type
Schools of Arts and Sciences
DUNS #
053599908
City
Corvallis
State
OR
Country
United States
Zip Code
97339
Lee, P; Hruby, D E (1995) Analysis of the role of the amino-terminal peptide of vaccinia virus structural protein precursors during proteolytic processing. Virology 207:229-33
Whitehead, S S; Bersani, N A; Hruby, D E (1995) Physical and molecular genetic analysis of the multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein. J Gen Virol 76 ( Pt 3):717-21
Lee, P; Hruby, D E (1994) Proteolytic cleavage of vaccinia virus virion proteins. Mutational analysis of the specificity determinants. J Biol Chem 269:8616-22
Vanslyke, J K; Hruby, D E (1994) Immunolocalization of vaccinia virus structural proteins during virion formation. Virology 198:624-35
Whitehead, S S; Hruby, D E (1994) Differential utilization of a conserved motif for the proteolytic maturation of vaccinia virus proteins. Virology 200:154-61
Whitehead, S S; Hruby, D E (1994) A transcriptionally controlled trans-processing assay: putative identification of a vaccinia virus-encoded proteinase which cleaves precursor protein P25K. J Virol 68:7603-8
Lee, P; Hruby, D E (1993) trans processing of vaccinia virus core proteins. J Virol 67:4252-63
Vanslyke, J K; Lee, P; Wilson, E M et al. (1993) Isolation and analysis of vaccinia virus previrions. Virus Genes 7:311-24
Vanslyke, J K; Whitehead, S S; Wilson, E M et al. (1991) The multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein: a degenerate conserved cleavage motif within core proteins. Virology 183:467-78
VanSlyke, J K; Franke, C A; Hruby, D E (1991) Proteolytic maturation of vaccinia virus core proteins: identification of a conserved motif at the N termini of the 4b and 25K virion proteins. J Gen Virol 72 ( Pt 2):411-6