Screening of the closterovirus genome by degenerate primer-mediated polymerase chain reaction.

Abstract

The genome of beet yellows virus (BYV), the type representative of the closterovirus group, encodes a homologue of the cellular heat-shock protein (HSP) 70 family. A pair of degenerate primers targeted to motifs A and E, which are highly conserved in HSP70s, was synthesized. Genomes of several definite and possible members of the closterovirus group were screened for the presence of the HSP70 gene with PCR using these degenerate primers. BYV, citrus tristeza virus (CTV), beet yellow stunt virus (BYSV) and carnation necrotic fleck virus templates produced 1 kb amplification products, which were shown by sequencing to represent fragments of the respective HSP70 genes. Further screening was performed with an additional degenerate primer targeted to the motif IV of the putative viral polymerase. This degenerate primer and specific primers complementary to the 5' region of the HSP70 genes of the respective viruses were used to estimate the distance between polymerase motif 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 regions of CTV (3026 nucleotides) and BYSV (2837 nucleotides) were cloned and sequenced. CTV and BYSV were found to encode the gene for an additional 30K (BYSV) or 33K (CTV) protein between the polymerase and the small hydrophobic protein genes, which was absent in BYV. These two 30K proteins displayed very weak similarity to each other, unlike the highly conserved polymerases, hydrophobic proteins and HSP70s of BYV, CTV and BYSV. Degenerate primer-mediated PCR proved to be an efficient tool for rapid screening and subsequent cloning of the viral genomes.