Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-1-encoded polyprotein.
Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250K polyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.
Hemmer
O
O
Institut de Biologie Moléculaire des Plantes, CNRS, Université Louis Pasteur, Strasbourg, France.
Greif
C
C
Dufourcq
P
P
Reinbolt
J
J
Fritsch
C
C
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017860:Nepovirus / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics*; D011817:Rabbits; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D011506:Proteins; D012367:RNA, Viral; D014764:Viral Proteins; D010450:Endopeptidases