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 12394 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 1a) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein. The downstream ORF, 1b, overlaps ORF 1a and lacks an initiation codon. This ORF encodes an RNA-dependent RNA polymerase of M(r) 59276. 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 59500. ORF 4 encodes a protein with an M(r) of 54648 that shows similarity to the corresponding proteins of other closteroviruses. ORF 5 encodes the viral coat protein (CP) with an M(r) of 35416. 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 55805 and 50164, respectively. ORFs 8 and 9 encode proteins of M(r) 21558 and 23771 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.