Nucleotide sequence and organization of ten open reading frames in the genome of Grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs

The genome of Grapevine leafroll-associated virus 1 (GLRaV-1) was cloned and the sequence of 12 394 nts determined. It contains 10 major open reading frames (ORFs) and a 3'-non-coding region lacking a poly(A) tract. The first ORF (ORF la) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein, The downstream ORF, 1 b, overlaps ORF la and lacks an initiation codon, This ORF encodes an RNA-dependent RNA polymerase of M-r 59 276, ORF 2 encodes a small hydrophobic protein of M-r 6736, and ORF 3 encodes a homologue of the HSP70 family of heat shock proteins and has an M-r of 59 500, ORF 4 encodes a protein with an M-r of 54 648 that shows similarity to the corresponding proteins of other closteroviruses, ORF 5 encodes the viral coat protein (CP) with an M-r of 35 416, The identity of this ORF as the CP gene was confirmed by expression in Escherichia coli and testing with the viral antibody. ORFs 6 and 7 code for two CP-related products with M-r of 55 805 and 50 164, respectively. ORFs 8 and 9 encode proteins of M-r 21 558 and 23 771 with unknown functions. Using DNA probes to different regions of the GLRaV-1 sequence, three major 3'-coterminal subgenomic RNA species were identified and mapped on the GLRaV-1 genome, Phylogenetic analyses of the individual genes of GLRaV-1 demonstrated a closer relationship between GLRaV-1 and GLRaV-3 than with other closteroviruses.