Nucleotide sequence and infectious cDNA clone of the L1 isolate of Pea seed-borne mosaic potyvirus

• Published in: Archives of virology 2001-01, Vol.146 (1), p.15-25
• Main Authors: OLSEN, B. S, JOHANSEN, I. E
• Format: Article
• Language: English
• Subjects: Bacteriology; Base Sequence; Biological and medical sciences; Carcinogenesis, carcinogens and anticarcinogens; Chenopodiaceae - microbiology; Cloning; Cloning, Molecular; Computer applications; Cultivars; DNA, Complementary - genetics; E coli; Epidemiology; Escherichia coli; Escherichia coli - genetics; Fjords; Fundamental and applied biological sciences. Psychology; Genome, Viral; Genomes; Genotypes; Inoculation; Introns; Medical sciences; Microbiology; Molecular Sequence Data; Nucleotide sequence; Open Reading Frames; Pea seed-borne mosaic potyvirus; Pea seed-borne mosaic virus; Pisum; Pisum sativum - virology; Plant Diseases - virology; Plant viruses; Plasmids; Polyadenylation; Potyvirus; Potyvirus - genetics; Proteolysis; Transfection; Tumors; Viruses
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s007050170187

The complete nucleotide sequence of Pea seed-borne mosaic potyvirus isolate L1 has been determined from cloned virus cDNA. The PSbMV L1 genome is 9895 nucleotides in length excluding the poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9594 nucleotides. The ORF potentially encodes a polyprotein of 3198 amino acids with a deduced Mr of 363537. Nine putative proteolytic cleavage sites were identified by analogy to consensus sequences and genome arrangement in other potyviruses. Two full-length cDNA clones, p35S-L1-4 and p35S-L1-5, were assembled under control of an enhanced 35S promoter and nopaline synthase terminator. Clone p35S-L1-4 was constructed with four introns and p35S-L1-5 with five introns inserted in the cDNA. Clone p35S-L1-4 was unstable in Escherichia coli often resulting in amplification of plasmids with deletions. Clone p35S-L1-5 was stable and apparently less toxic to Escherichia coli resulting in larger bacterial colonies and higher plasmid yield. Both clones were infectious upon mechanical inoculation of plasmid DNA on susceptible pea cultivars Fjord, Scout, and Brutus. Eight pea genotypes resistant to L1 virus were also resistant to the cDNA derived L1 virus. Both native PSbMV L1 and the cDNA derived virus infected Chenopodium quinoa systemically giving rise to characteristic necrotic lesions on uninoculated leaves.