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 c lones 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 t hese clones to be replicated was investigated by protoplast infection in the presence of a helper virus. We demonstrate that the 5' and 3' n on-coding regions as well as the satellite-encoded P3 protein are esse ntial for replication of the GFLV-F13 satellite RNA. Our results sugge st that two hydrophobic regions located at the N- and C-extremity of P 3 and a zinc-finger motif near the C-terminal extremity of P3 are prob ably involved in the replication of this satellite. Analysis of the in vitro translation products from transcripts of RNA3 clones of differe nt lengths indicates that the double band formed by P3 could result fr om phosphorylation of a part of this protein.