Downloaded from www.microbiologyresearch.org by IP: 54.234.201.175 On: Thu, 18 May 2017 05:07:07 Journal of General Virology (1991), 72, 2625-2632. Printed in Great Britain 2625 The complete nucleotide sequence of pea seed-borne mosaic virus RNA E. Johansen, 10. F. Rasmussen, 2 M. Heide 3 and B. Borkhardt 1. 1The Danish Research Service for Plant and Soil Science, Lottenborgvej 2, DK-2800 Lyngby, 2Genetic Engineering Group, Building 227, The Technical University of Denmark, DK-2800 Lyngby and 3plant Protection Division, Novo Nordisk A/S, Novo all~, DK-2880 Bagsvcerd, Denmark The complete nucleotide sequence of the RNA genome of pea seed-borne mosaic virus (PSbMV) was deter- mined from cloned cDNA and by direct sequencing of viral RNA. The PSbMV genomic sequence was determined to be 9924 nucleotides in length excluding the poly(A) tract. The RNA contained an open readiog frame (ORF) of 9618 nucleotides with the potential to encode a polyprotein with a calculated Mr of 364000 (364K). The ORF was flanked by a 5' untranslated leader sequence of 143 nucleotides and a 3' untranslat- ed region of 163 nucleotides. A comparison of the PSbMV polyprotein with the polyproteins of the potyviruses tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV) and potato virus Y (PVY) showed that PSbMV had a similar genome organization. The polyproteins had a high level of amino acid identity except in the N- terminal region, which varied in both sequence and length. Putative proteolytic cleavage sites were identi- fied in the polyprotein of PSbMV by comparison with those identified for other potyviruses. The cleavage site between the 6K protein and the 49K proteinase is proposed to occur at the C-terminal side of glutamic acid and not at the C-terminal side of glutamine as in other potyviruses. In addition to the five proteolytic cleavage sites for the 49K proteinase identified pre- viously, a sixth putative cleavage site was identified internally in the 49K proteinase of PSbMV, as well as in the 49K proteinases of TEV, TVMV, PPV, PVY and soybean mosaic virus. Cleavage at this site in the 49K proteinases of TEV, TVMV and PPV would result in an N-terminal protein of 22K to 24K, which is similar in size to the size determined for their VPgs. Introduction Pea seed-borne mosaic virus (PSbMV) is a member of the potyvirus group. The flexuous, rod-shaped particles of the potyviruses contain a positive-sense ssRNA genome of approximately 10 kb (Hollings & Brunt, 1981). The 5' terminus of the RNA is covalently linked to a virus-encoded protein (VPg) (Murphy et al., 1990) and the 3' end terminates with a poly(A) tract (Hari et al., 1979). The complete nucleotide sequences of the potyviruses tobacco etch virus (TEV; Allison et al., 1986), tobacco vein mottling virus (TVMV; Domier et al., 1986), plum pox virus (PPV; Maiss et al., 1989) and potato virus Y (PVY; Robaglia et al., 1989) have been determined. Analysis of these sequences has demonstrated that the potyvirus genome contains one large open reading frame (ORF) with the potential to encode a polyprotein of between 3005 amino acids (TVMV) and 3063 amino acids (PVY). The polyprotein is proteolytically processed into at least eight proteins (Carrington & Dougherty, 1987 a, b; Carrington et al., 1989, 1990): the N-terminal protein, the helper component-proteinase (HC-Pro), a 42K to 50K protein, the cytoplasmic inclusion protein (CI), a 6K protein, the small nuclear inclusion protein (NIa), the large nuclear inclusion protein (NIb) and the coat protein (CP) (Dougherty & Carrington, 1988). The functions of some of the non-structural proteins have been established. HC-Pro is necessary for aphid trans- mission (Thornbury et aL, 1985) and also has proteolytic activity (Carrington et al., 1989); CI has helicase activity (Lain et al., 1990); NI a, or the N-terminal part of it, is VPg, which is covalently linked to the RNA (Murphy et al., 1990; Riechmann et al., 1989). NI~ also has proteolytic activity (Carrington & Dougherty, 1987a) and will be referred to as the 49K proteinase (49K-Pro). NIb is expected to have RNA polymerase activity owing to its homology with other viral RNA-dependent RNA polymerases (Domier et al., 1987). We have determined the complete nucleotide se- quence of the PSbMV genome. Analysis of the deduced amino acid sequences of PSbMV, TEV, TVMV, PPV, PVY and soybean mosaic virus (SMV) suggests the presence of an additional proteolytic cleavage site in 49K-Pro. 0001-0296 © 1991 SGM