Inhibition of protein synthesis by vaccinia virus I. Characterization of an inhibited cell-free protein-synthesizing system from infected Cells

Abstract

L-929 or HeLa cells infected with vaccinia virus in the presence of cycloheximide fail to resume protein synthesis upon removal of the drug 3.5 hr after infection. However, infected-treated LLC-MK2 cells resume protein synthesis upon removal of the drug. Cell-free protein-synthesizing systems prepared from such vaccinia virus infected-cycloheximide treated L-929 or HeLa cells, 30 min after removal of the drug, fail to incorporate amino acids in vitro in response to endogenous mRNAs, while similar extracts of LLC-MK2 cells are functional in vitro. The inhibition of protein synthesis is also characterized by a failure to respond to exogenous mRNAs (Globin, L cell poly (A), or EMC RNA). Such inhibited extracts have been separated into supernatant, ribosome, and ribosomal salt wash fractions. The response of ribosomes from infected and uninfected cells to EMC RNA in the presence of homologous and heterologous supernatant fractions shows that defects can be found in both ribosomes and supernatant fractions of infected-treated cells. Readdition of salt wash fraction of infected and uninfected ribosomes to the ribosome and supernatant fractions of infected and uninfected cells shows an additional lesion in the salt wash fraction of infected-treated cells. Addition of supernatant, ribosome, and salt wash fractions of infected and uninfected cells individually to reconstituted protein-synthesizing systems prepared from normal cells shows that the primary lesion in the infected-treated cells is the salt wash fraction of the infected ribosomes. This constitutes a crude initiation factor preparation and is consistent with previous observations that vaccinia virus-induced inhibition of protein synthesis is the result of a failure at the level of initiation.