Hypervariable genes in Grapevine leafroll associated virus 1.

Analysis of nucleotide sequences of 10 open reading frames from the Grapevine leafroll associated virus 1 (GLRaV-1), a tentative member of the genus Closterovirus, revealed the presence of an unusually high degree of sequence variation in ORFs 3, 6 and 7 encoding a homologue of heat shock protein 70 and two diverged copies of the coat protein (CPd1 and CPd2), respectively. Overall, 75 clones corresponding to ORFs 3, 6 and 7 were sequenced and 1916 nucleotide changes were recorded relative to the published sequence. Surprisingly, none of the changes resulted in a frame shift or stop codon and there was a trend for the conservation of amino acids or change to amino acids having similar physiochemical properties. The CPd2 gene was particularly variable with a mutation seen in 60% of the nucleotide positions in one or more of the 1.1-kb cDNA clones sequenced. These observations suggest that GLRaV-1 may exist in the form of a heterogeneous population, possibly resulting from the lack of selective pressure and from mixing of virus strains due to viticulture practices of vegetative propagation and grafting over the centuries.

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.

Efficient cloning of cDNA from grapevine leafroll-associated virus 4 and demonstration of probe specificity by the viral antibody.

Using a random-PCR method, a cDNA clone (LR4) was constructed from the replicative form dsRNA of grapevine leafroll-associated virus 4 (GLRaV-4). Northern blot analysis showed hybridization of LR4 to dsRNA in an extract of a Thompson Seedless grapevine clone from which GLRaV-4 was isolated originally by Hu et al. (1990). The cDNA clone was sequenced and shown to be specific to GLRaV-4 by reverse-transcription-PCR using GLRaV-4 particles enriched by the virus antibody coupled to magnetic beads. Reverse-transcription-PCR was used successfully to screen different varieties of grapevines for the virus. Western blot analysis of GLRaV-4 extracts from different varieties of infected grapevines revealed two distinct species of capsid protein with estimated Mr of either 35500 or 38000 depending on the variety used. Both proteins reacted with polyclonal as well as monoclonal antibodies.