The mechanism of vaccinia virus-induced inhibition of protein synthesis was studied in LLC-MK2, HeLa and L cells. Removal of cycloheximide (300 µg/ml) from cells infected at a multiplicity of infection (m.o.i.) of 300 particles/cell at 4 h after infection resulted in the resumption of both host and virus protein synthesis in LLC-MK2 cells, but not in HeLa and L cells. In order to determine whether virus-induced RNA synthesis, which occurs in infected cells in the presence 01 cycloheximide, is related to the inhibition of protein synthesis, (cut-off), the rate of virus-induced RNA synthesis in the presence of cycloheximide was measured in all three cell types. In L cells and HeLa cells the rate of virus-induced RNA synthesis increased with time, whereas in LLC-MK2 cells it remained constant for at least 4 h. However, when higher multiplicities (900 and 2700 particles/cell) were used to infect LLC-MK2 cells, the rate of RNA synthesis in the presence of cycloheximide did increase with time and was greater at the higher multiplicity. Under these conditions there was a direct relationship between the extent of virus RNA synthesis and the degree of cut-off after the removal of cycloheximide. In HeLa and L cells infected at 300 particles/cell, the longer the exposure to cycloheximide, the greater was the cut-off observed upon removal of the drug. As was the case with LLC-MK2 cells, there was a direct relationship between the rate of RNA synthesis and the degree of inhibition of protein synthesis. Since virus-induced RNA synthesis occurs in the presence of cycloheximide, the effects of actinomycin D and cordycepin on host polypeptide synthesis were tested. Inhibition of host cell protein synthesis was virtually abolished when HeLa cells were infected in the presence of cordycepin (50 and 25 µg/ml) or actinomycin D (20 µg/ml).

These results indicate that, as the rate of virus-induced RNA synthesis increased, regardless of the type of cell used, protein synthesis was inhibited at earlier times and to a greater extent. These observations are consistent with the hypothesis that the cut-off phenomenon is related to the synthesis of an early virus-induced RNA (s).


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