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

Abstract

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.