SCREENING OF THE CLOSTEROVIRUS GENOME BY DEGENERATE PRIMER-MEDIATED POLYMERASE CHAIN-REACTION

The genome of beet yellows virus (BYV), the type representative of the closterovirus group, encodes a homologue of the cellular heat-shock p rotein (HSP) 70 family. A pair of degenerate primers targeted to motif s A and E, which are highly conserved in HSP70s, was synthesized. Geno mes of several definite and possible members of the closterovirus grou p were screened for the presence of the HSP70 gene with PCR using thes e degenerate primers. BYV, citrus tristeza virus (CTV), beet yellow st unt virus (BYSV) and carnation necrotic fleck virus templates produced 1 kb amplification products, which were shown by sequencing to repres ent fragments of the respective HSP70 genes. Further screening was per formed with an additional degenerate primer targeted to the motif IV o f the putative viral polymerase. This degenerate primer and specific p rimers complementary to the 5' region of the HSP70 genes of the respec tive viruses were used to estimate the distance between polymerase mot if IV and the start point of the HSP70 gene for BYV (approximately 1.1 kb), CTV and BYSV (around 2.0 kb) by PCR. The amplified genome region s of CTV (3026 nucleotides) and BYSV (2837 nucleotides) were cloned an d sequenced. CTV and BYSV were found to encode the gene for an additio nal 30K (BYSV) or 33K (CTV) protein between the polymerase and the sma ll hydrophobic protein genes, which was absent in BYV. These two 30K p roteins displayed very weak similarity to each other, unlike the highl y conserved polymerases, hydrophobic proteins and HSP70s of BYV, CTV a nd BYSV. Degenerate primer-mediated PCR proved to be an efficient tool for rapid screening and subsequent cloning of the viral genomes.