LOCATION OF THE REPLICATION DETERMINANTS OF THE SATELLITE RNA ASSOCIATED WITH GRAPEVINE FANLEAF-NEPOVIRUS (STRAIN-F13)

A large satellite RNA of 1114 nucleotides, named RNA3, is always found associated with the genomic RNAs of grapevine fanleaf virus, isolate F13 (GFLV-F13). RNA3 encodes a non-structural protein (P3) of M(r) 37K to which no function has previously been assigned. Full-length cDNA clones of RNA3 were mutated in the 5' and 3' non-coding regions and in the 37K open reading frame. The ability of transcripts obtained from these clones to be replicated was investigated by protoplast infection in the presence of a helper virus. We demonstrate that the 5' and 3' non-coding regions as well as the satellite-encoded P3 protein are essential for replication of the GFLV-F13 satellite RNA. Our results suggest that two hydrophobic regions located at the N- and C-extremity of P3 and a zinc-finger motif near the C-terminal extremity of P3 are probably involved in the replication of this satellite. Analysis of the in vitro translation products from transcripts of RNA3 clones of different lengths indicates that the double band formed by P3 could result from phosphorylation of a part of this protein.