Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase.
The 49-kDa proteinase of tobacco etch virus (TEV) cleaves the polyprotein derived from the TEV genomic RNA at five locations. Molecular genetic and biochemical analyses of the 49-kDa TEV proteinase were performed to test its homology to the cellular trypsin-like serine proteases. A cDNA fragment, containing the TEV 49-kDa proteinase gene and flanking sequences, was expressed in a cell-free transcription/translation system and resulted in the formation of a polyprotein precursor that underwent rapid self-processing. Site-directed mutagenesis was used to test the effect of altering individual 49-kDa amino acid residues on proteolysis. The data suggest that the catalytic triad of the TEV 49-kDa proteinase could be composed of the His234, Asp269, and Cys339. These findings are consistent with the hypothesis that the TEV 49-kDa proteinase is structurally similar to the trypsin-like family of serine proteinases with the substitution of Cys339 as the active site nucleophile. A structural model of the TEV 49-kDa proteinase proposes other virus-specific differences in the vicinity of the active site triad and substrate-binding pocket. The structure may explain the observed negligible effect of most cellular proteinase inhibitors on the activity of this viral proteinase.
Dougherty
W G
WG
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Parks
T D
TD
Cary
S M
SM
Bazan
J F
JF
Fletterick
R J
RJ
D007611:Aprotinin / Q000494:pharmacology; D001665:Binding Sites; D004252:DNA Mutational Analysis; D005814:Genes, Viral; D007976:Leupeptins / Q000494:pharmacology; D010206:Papain / Q000037:antagonists & inhibitors; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010942:Plant Viruses / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics; D015032:Zinc / Q000494:pharmacology
D007976:Leupeptins; D014764:Viral Proteins; D007611:Aprotinin; D010447:Peptide Hydrolases; D010206:Papain; D015032:Zinc; C032854:leupeptin