Capsid Polypeptides in a Group III Virus from Gaeumannomyces graminis var. tritici Are Related

Abstract

SUMMARYPeptide mapping and amino acid analysis have shown that the two capsid polypeptides of a group III virus, 87-1-H, from Gaeumannomyces graminis are closely related.Isometric double-stranded RNA (dsRNA) virus particles are common in field isolates of the wheat take-all fungus, Gaeumannomyces graminis var. tritici (Ggt), and have been divided into four groups based on their physical and serological properties and the number and sizes of their dsRNA segments and capsid polypeptides (Buck et al., 1981;McFadden et al., 1983;Buck, 1984). Viruses in groups I and II have a divided genome of two dsRNA segments and have been placed in the family Partitiviridae (Brown, 1986). A recently described group III virus, designated 87-1-H, which has an undivided dsRNA genome of mol. wt. 4.2 x 106 and two capsid polypeptides, P1 and P2, mol. wt. 84000 and 78000 (Jamil & Buck, 1984), is a possible member of the family Totiviridae (Brown, 1986). However, viruses in the Totiviridae have only a single capsid polypeptide. To determine whether the 87-1-H capsid polypeptides are unrelated or whether one could be derived-from the other we have compared them by peptide mapping and by determining their amino acid compositions.Ggt isolate 87-1 was cultured and virus 87-1-H was isolated and purifiedas described by Jamil & Buck (1984). For the separation of capsid polypeptides P 1 and P2, a virus suspension was first made 19/0 in SDS and 0.1% in 2-mercaptoethanol and heated to 100 °C for 3 min. The denatured capsid polypeptides were then separated by polyacrylamide gel electrophoresis (PAGE) at 5 V/cm for 24 h using a 4% stacking gel, a 10% resolving gel and the Laemmli (1970) discontinuous buffer system. The gel was stained for 15 min with 0.1% Coomassie Brilliant Blue in 50% (v/v) methanol with 10% (v/v) acetic acid and then destained for 1 h in 5% (v/v) methanol with 10 ~o (v/v) acetic acid. A typical separation is shown in Fig. 1. Individual protein bands were excised, further purified by a second cycle of PAGE using 16~o resolving gels and then electroeluted (Leibowitz & Wang, 1984) into dialysis bags containing 0.05 M-Tris-HCI, 0-24 M-glycine, 0"1% SDS, pH 8.3. The solutions were adjusted to 0.2 M-KCI and stored at 0:°C for 15 min. The precipitated polypeptides were collected by centrifugation, washed with 0.1 M-HC1 in acetone and then with acetone, and then dried in vacuo. Analysis by SDS-PAGE with Coomassie Brilliant Blue staining showed that each polypeptide was free from detectable contamination by the other.For peptide mapping, polypeptides (5 to 10 p.g) were dissolved in 0.5 M-sodium phosphate, 0.19/0 SDS, pH 7.5 and iodinated with 12s I using Iodogen (Pierce Chemical Company, Rockford, IlL, U.S.A.) as described by Fraker & Speck (1978). Iodinated polypeptides were digested at 37 °C for 19 h either with trypsin (100 ~tg/ml) in 0.1 M-ammonium bicarbonate, or with pepsin (100 ~tg/ml) in 5 % (v/v) formic acid, then diluted fivefold with water and lyophilized. Peptides t Present address:

The Capsid Polypeptides of the 190S Virus of Helminthosporium Victoriae

“…The group III virus, designated 87-1-H, from Gaeumannomyces graminis (Jamil & Buck, 1986) and the virus from the yeast Yarrowia lipolytica (EI-Sherbeini et al, 1987) have recently been reported to possess two or more related capsid polypeptides of comparable size to those of the 190S virus. The larger of the two capsid polypeptides of G. graminis 87-1-H virus, unlike p88 of H. victoriae 190S virus, appears to convert to the smaller polypeptide when virus preparations are stored at 4 °C for 1 month or longer (Jamil & Buck, 1986).…”