Inhibition of protein synthesis by vaccinia virus: IV. The role of low-molecular-weight viral RNA in the inhibition of protein synthesis

The role of viral RNA in the inhibition of protein synthesis (shutoff) was studied in HeLa cells infected with uv-irradiated vaccinia virus. Increasing doses of uv irradiation inactivate the transcriptional activity of vaccinia virus with single-hit kinetics, as analyzed in vitro with activated viral cores and in vivo, with rapidly labeled cytoplasmic RNA. The size of the RNA synthesized in vitro and in vivo is found to decrease with increasing doses of uv irradiation. Under conditions where protein synthesis is still inhibited, a size of 50–100 nucleotides is observed in the cytoplasm of cells infected with virus which had been uv irradiated to 9600 ergs/mm 2. Southern hybridization of 32P-labeled cytoplasmic RNA to EcoRI restriction fragments of vaccinia DNA show that virus-specific sequences are transcribed when cells are infected with various preparations of virus uv irradiated up to 9600 ergs/mm 2. No hybridization to EcoRI DNA restriction fragments is detected with RNA obtained from cells infected with higher doses of uv irradiation (17,280 and 28,800 ergs/mm 2). When cells infected with uv-irradiated virus capable of causing shutoff are treated with high doses of actinomycin D (20 and 30 μg/ml) inhibition of protein synthesis is prevented. Lower doses of actinomycin D (2, 5, and 10 μg/ml) only partially prevent this inhibition. Actinomycin D at 30 μg/ml has no effect on primary uncoating of uv-irradiated virus (9600 ergs/mm 2). These results suggest that low-molecular-weight viral RNA (50–100 nucleotides) is associated with shutoff.