Summary auto-generated
This study determined the complete 5834-nucleotide sequence of RNA 1 from beet soil-borne furovirus (BSBV, Ahlum isolate) using a novel approach. Since BSBV is difficult to purify and no prior cDNA clones existed for RNA 1, researchers designed primers targeting highly conserved helicase-encoding regions found in related furo-, hordei-, and tobraviruses. These primers successfully amplified a 239-nucleotide PCR product that served as a starting sequence. Using RT-PCR with specific and random primers, the complete RNA 1 sequence was obtained. The analysis revealed BSBV RNA 1 contains a single large open reading frame encoding proteins of 145 kDa and 204 kDa through stop codon readthrough. The N- and C-terminal regions contain conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs typical of plant virus replicases. Notably, unlike other related viruses, BSBV RNA 1 lacks additional genes downstream of the replicase-coding region. The 3' untranslated region contains a tRNA-like structure similar to those found on BSBV RNAs 2 and 3.
Key findings
- Complete sequencing of BSBV RNA 1 (5834 nucleotides) was achieved using primers designed from conserved helicase domains of related viruses as a starting point for amplification
- BSBV RNA 1 encodes a single large open reading frame producing a 145 kDa protein and a 204 kDa readthrough protein containing conserved replicase motifs (methyltransferase, helicase, RNA-dependent RNA polymerase)
- BSBV RNA 1 lacks additional genes found in related furoviruses and tobraviruses, distinguishing its genome organization
- The 3' terminus of RNA 1 contains a tRNA-like structure with a valine anticodon (GAC) that differs from RNAs 2 and 3 (CAC anticodon)
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Abstract
The complete sequence of the 5834 nucleotides of RNA 1 of beet soil- borne furovirus (BSBV, Ahlum isolate) was determined using a PCR product obtained with primers to highly conserved coding regions for helicase-like proteins in RNA 1 of furo-, hordei- and tobraviruses as a starting sequence. Unknown parts of the sequence upstream and downstream of this starting sequence were amplified by means of RT-PCR techniques using combinations of specific and random primers. BSBV RNA 1 contains one large ORF for a readthrough protein with a molecular mass of 204 kDa (204K protein) which is interrupted internally by a UAA stop codon terminating the coding region for a protein of 145 kDa (145K protein). The N- and C-terminal parts of the 145K protein and the readthrough domain of the 204K protein contain methyltransferase, helicase and RNA-dependent RNA polymerase motifs, respectively. Unlike other furo- and tobraviruses BSBV contains no further genes on its RNA 1.