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

CSIRO Plant Industry and Cooperative Research Center for Viticulture, Adelaide Laboratory, PO Box 350, Glen Osmond, South Australia 5064 1 Author for correspondence: Ali Rezaian. Fax +61 8 8303 8601. e-mail ali.rezaian{at}pi.csiro.au 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.