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

Abstract

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.