Topology of the major capsid protein P3 of bacteriophage PRD1: analysis using monoclonal antibodies and C-terminally truncated proteins.
Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid-protein vesicle containing the viral dsDNA genome. In this study we characterize a panel of monoclonal antibodies (MAb) against P3. The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules. Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions. Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies. Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein. The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid. MAbs 3N81 and 3R2 recognize epitopes in the region of amino acids 159-168, which is part of the first predicted beta-barrel structure of P3. The third antigenic region is in the second predicted beta-barrel, between amino acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map. The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17. Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation.
Bamford
J K
JK
Department of Genetics, University of Helsinki, Finland.
Luo
C
C
Juuti
J T
JT
Olkkonen
V M
VM
Bamford
D H
DH
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000918:Antibody Specificity; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D000939:Epitopes; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000276:immunology; D017384:Sequence Deletion
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins; D000939:Epitopes; D011994:Recombinant Proteins; C084392:protein P3, bacteriophage PRD1