Cleavage of potato virus Y polyprotein in Escherichia coli depending on the proteolytic activity of viral protease.
We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus. Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome. To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli. By Western blotting analysis of the cell extract of E. coli expressing the polyprotein, mature CP was detected. A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein. These results indicated that the polyprotein expressed in E. coli was cleaved depending on the proteolytic activity of PVY protease.
Hidaka
M
M
Department of Biotechnology, Faculty of Agriculture, University of Tokyo, Japan.
Yoshida
Y
Y
Masaki
H
H
Uozumi
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005814:Genes, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D011498:Protein Precursors; D011506:Proteins; D014764:Viral Proteins; D004247:DNA; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses