Tobacco protoplasts inoculated with pea enation mosaic virus (PEMV) contained virus antigen which was detectable by fluorescent antibody staining 15 h or more after inoculation. Increase in the proportion of fluorescing protoplasts and in the intensity of fluorescence with time of culture suggested that new virus antigen was synthesized. At early stages the fluorescence was found both in the nucleus and in the cytoplasm, but by about 48 h it was more restricted to the nucleus. The uptake of [P] into virus and into virus RNA indicated that PEMV multiplied in the protoplasts, but the yield of virus was relatively low, about 1 to 5 × 10 particles per infected protoplast. The optimum inoculation conditions for routine infection as assessed by fluorescent antibody staining were 1.5 µg/ml PEMV, 1.5 µg/ml poly--ornithine, and pH 4.7. Protoplasts could be infected in the absence of poly--ornithine at the higher PEMV concentrations. Actinomycin D added to the culture medium reduced the number of fluorescing protoplasts and the intensity of fluorescence.


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