The nucleotide sequence of the genomic RNA 1, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-FI 3) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an Mr of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picornavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-FI3 RNAI. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the snbstrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of Mr 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease eofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.

Transcripts were produced in vitro by run-off transcription from full-length eDNA of RNA1 and RNA2 of grapevine fanleaf nepovirus (GFLV; isolate F13) cloned downstream from a bacteriophage RNA polymerase promoter. These transcripts, which possess a 5' terminal cap structure and a non-viral G residue instead of the naturally occurring genome-linked viral protein (VPg), are infectious to Chenopodium quinoa protoplasts when inoculated by electroporation. Synthetic RNA1 alone replicated in protoplasts. Inoculation of C. quinoa plants with synthetic RNA1 plus RNA2 produced symptoms similar to, but weaker, than those observed in plants infected with natural GFLV 6 to 8 days post-inoculation. Co-inoculated RNA1 and RNA2 were able to replicate and spread systemically in plants but RNA1 alone produced no symptoms and was not detected in noninoculated leaves, suggesting that virus spread requires RNA2. Analysis of the genomic RNAs in plants infected with transcripts showed that the non-viral G at their 5' ends was not retained in the progeny.

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