Generation and characterization of monoclonal antibodies reactive with the 49-kDa proteinase of tobacco etch virus.
Monoclonal antibodies (McAbs) were generated against two tobacco etch virus (TEV)-encoded nonstructural proteins, the 49-kilodalton (kDa) proteinase and the 58-kDa putative RNA-dependent RNA polymerase. This process was facilitated by the fact that these two TEV nonstructural proteins cocrystallize in the nuclei of virus-infected cells to form nuclear inclusion (NI) bodies which can be purified readily. The anti-NI McAbs were shown by Western blot analysis to be specific for either the TEV 49-kDa or the 58-kDa protein. Those McAbs reactive with the 49-kDa proteinase were characterized further with respect to the 49-kDa domain with which they reacted and with respect to their ability to inhibit the autocatalytic or self-processing activity of the 49-kDa proteinase. The 49-kDa antigens were synthesized from a TEV cDNA sequence using cell-free transcription and translation systems. Each anti-49-kDa McAb was used in immunoprecipitation studies with a series of 49-kDa antigens which represented a nested set of 49-kDa proteins with common amino termini but varying in length. Immunoprecipitation results showed that all of the anti-49-kDa proteinase McAbs reacted with one of five binding regions, designated A through E from the carboxy terminus of the proteinase, which were 77, 38, 81, 18, and 61 amino acids long, respectively. The 38-amino-acid binding region B contained the proposed catalytic cysteine 339 residue and was recognized by only one McAb, 4911. McAb 4911 was the only anti-49-kDa McAb capable of inhibiting the self-processing reaction in which the 49-kDa proteinase is released from its 75-kDa polyprotein precursor.
Slade
D E
DE
Department of Microbiology, North Carolina State University, Raleigh 27695.
Johnston
R E
RE
Dougherty
W G
WG
D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D010450:Endopeptidases / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas; D010942:Plant Viruses / Q000201:enzymology* / Q000276:immunology; D010957:Plasmids; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D010450:Endopeptidases