Autocatalytic activity of the tobacco etch virus NIa proteinase in viral and foreign protein sequences.
The small nuclear inclusion (NIa) protein of the tobacco etch virus (TEV) is synthesized initially as part of a genome-derived high M(r) precursor. The NIa protein releases itself from this genome-derived precursor by self-cleavage, or an autocatalytic processing event. Cleavage between specific glutamine-glycine dipeptides at the N and C termini generates the 430 amino acid or 49,000 M(r) (49K) NIa protein. The requirements of this autocatalytic release, or cis cleavage, were examined by constructing gene cassettes encoding the TEV NIa protein which could be ligated into particular locations in cDNA of the TEV genome and also into foreign gene DNA sequences. Using cell-free transcription and translation systems, polyproteins containing TEV NIa sequences were synthesized and assayed for (i) autocatalysis and (ii) the ability of a functional NIa proteinase, purified from plant tissue, to cleave in bimolecular or trans reactions various artificial polyproteins which contained an inactive form of the NIa proteinase. The NIa self-cleavage events required an active proteinase sequence and a consensus TEV cleavage site sequence at the N and C termini. These results were consistent for NIa protein sequences placed at a foreign TEV cleavage site or in unrelated proteins. Differences were noted in the trans cleavage of these sites.
Rorrer
K
K
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Parks
T D
TD
Scheffler
B
B
Bevan
M
M
Dougherty
W G
WG
D000595:Amino Acid Sequence; D002384:Catalysis; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics; D010947:Plants, Toxic; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000382:microbiology*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses