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P isolate of tomato spotted wilt virus (TSWV-P) was purified by ultracentrifugation followed by metrizamide isopycnic density-gradient. Nucleocapsid purified after Triton X-100 treatment was disrupted with 1% SDS and analyzed by 1% agarose gel electrophoresis. RNA patterns were indistinguishable from those obtained from intact particles and both contained six single-stranded RNA species (ss1-ss6). When RNAs were extracted by phenol and precipitated by ethanol, three double-stranded RNA species (ds1-ds3) were appeared. The ds1, ds2 and ds3 was formed after annealing ss1 with ss2, ss3 with ss4 and ss5 with ss6, respectively. Glyoxal treatment rendered ds1-ds3 to three single-stranded RNA segments (L, M and S RNA). In Northern blot hybridization analyses using three glyoxalated single-stranded RNA segments as probes, S RNA hybridized with ss5 and ss6, in addition to ds3. M RNA hybridized with ss3, ss4 and ds2. L RNA also hybridized with ss1, ss2 and ds1. Moreover, S RNA segment, ss5, ss6 and ds3, hybridized with oligonu-cleotide probes of both polarities representing 3′ end sequence of S RNA segment and an internal part on N protein coding region. A probe of viral strand on N protein coding region, however, hybridized weakly to S RNA segment and seemed not to react to ss5. Interestingly, the amount of viral strand was about 4 times larger than the amount of viral complementary strand in S RNA species, as judged from reactivity of hybridization using strand-specific probes. These results indicate that three different-sized RNA segments are present in both viral and viral complementary strands in the nucleocapsid of TSWV.

The Presence of Three Pairs of Possibly Complementary RNA Species in Isolated Nucleocapsid Material of Tomato Spotted Wilt Virus.