Seiko Imaizumi

Synthesis of single- and double-stranded cucumber mosaic virus RNAs in tobacco mesophyll protoplasts.

Five major RNAs were isolated by polyacrylamide gel electrophoresis from purified virions of the yellow strain of cucumber mosaic virus (CMV-Y), although the smallest RNA (RNA 5) was not detectable in RNAs of the ordinary strain of CMV (CMV-O). Synthesis of CMV-specific RNAs was investigated using tobacco mesophyll protoplasts inoculated with CMV in vitro . Incorporation of [ 3 H]uracil into RNAs was analyzed by electrophoresis in polyacrylamide gels. A combination of actinomycin D treatment and ultraviolet irradiation to protoplasts showed a marked inhibiting effect on host RNA synthesis, but little on viral RNA synthesis. Replication of the five major RNAs of CMV-Y occurred in the protoplasts, although RNAs 1 and 2 could not be resolved; RNAs 3 and 5 were especially predominant. In the case of CMV-O, RNA 5 was not synthesized in vivo . The infectivity of RNA extracted from CMV-infected tobacco protoplasts was detectable within 6 hr after inoculation, and reached its maximum at 18 hr; it then decreased rather sharply. The rate of viral RNA synthesis was highest at 12 to 18 hr after inoculation, and after that it gradually slowed. Double-stranded RNAs were also isolated from the infected protoplasts. Existence of five pairs of replicative form (RF) and replicative intermediate (RI) RNAs corresponding to the respective single-stranded CMV RNAs was assumed by examining their chromatographic behavior on a cellulose column, solubility in 1 M NaCl and susceptibility to ribonuclease treatment. The smallest RI, 5, was eluted from the cellulose column together with single-stranded RNAs with a buffer containing 15% ethanol. Much larger amounts of RF 3 and 5 were detected as compared with the other RF species, and the electrophoretic pattern of the RFs was very similar to that of single-stranded CMV RNAs syntheiszed in vivo except for the slower electrophoretic mobilities of the RFs.

Formation of anti-plant viral protein by Mirabilis jalapa L. cells in suspension culture

The extract of Mirabilis jalapa cultured cells and its precipitate fraction with 90% saturated ammonium sulfate showed an anti-plant viral activity comparable to that of the roots and leaves of the original plant. In the immunodiffusion experiment, the extract of cultured cells positively reacted with MAP (Mirabilis Anti-plant viral Protein) anti-serum. The changes in MAP formation during cell growth and the MAP content of roots and leaves were examined using enzyme-linked immunosorbent assay (ELISA). MAP formation proceeded almost in parallel with cell growth. The MAP content of cultured cells reached the highest level (0.6 mg/g dry weight) on the 9th day after inoculation, which was less than one-third of the content of the roots but three times larger than that of the leaves.

Identical 3′-termini of the four RNA species of cucumber mosaic virus

Abstract The 3′-ends of the four main RNA species isolated from purified cucumber mosaic virus were labeled with tritium by periodate oxidation, followed by reduction with [ 3 H]borohydride. Each RNA species, fractionated by polyacrylamide gel electrophoresis, was digested with alkali and the resultant 3′-termini were analyzed by phosphocellulose column chromatography. All of the four RNA species have a nonphosphorylated adenosine residue at their 3′-end.