Cloning and in vitro characterization of the grapevine fanleaf virus proteinase cistron.
The region of the genomic RNA-1 from grapevine fanleaf virus isolate F13 (GFLV-F13), containing the proteinase cistron and flanking sequences (nucleotides 3894 to 4789) of the GFLV polyprotein, was modified by PCR mutagenesis to create a start codon and cloned in a transcription vector. The transcripts from the resulting clone (pVP7) produced, upon translation in rabbit reticulocyte lysate, a 37.8-kDa protein which was subsequently cleaved to a stable 28-kDa product. Autocleavage was maximal at pH 7.0-8.5 and at 30 degrees. Inhibition of the activity was greater than 80% when translation was performed in the wheat germ system. In rabbit reticulocyte lysate, inhibition was also obtained with PMSF, EDTA, E-64, Ca+2, Zn+2, and Co+2. The pVP7 translation product acts in cis, in the case of its autocleavage, or in trans in the processing of the viral 122-kDa polyprotein from GFLV RNA-2 into a 66-kDa protein and the 56-kDa coat protein. The carboxy extremity of the complete pVP7 translation product, encoded by nucleotides 4633 to 4789 of RNA-1, was not required for the proteinase activity, at least in trans.
Margis
R
R
Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, Strasbourg, France.
Viry
M
M
Pinck
M
M
Pinck
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005796:Genes / Q000235:genetics*; D005814:Genes, Viral / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010942:Plant Viruses / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology; D012328:RNA Viruses / Q000235:genetics*; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D002413:Cations, Divalent; D011480:Protease Inhibitors; D014764:Viral Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C071903:GFLV VP7 proteinase