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.