Shut‐Off of Host Protein Synthesis in Vaccinia‐Virus‐Infected Cells Exposed to Cordycepin

Abstract

Cytoplasmic lysates from Ehrlich ascites tumor cells were prepared for very efficient endogenous protein synthesis in vitro. It was shown that initiation of polypeptide chain occurred in vitro since 70–85% of protein synthesis in vitro was inhibited by aurintricarboxylic acid or edeine, it was also calculated that about three protein chains were synthesized per active ribosome from the amount of [35S]methionine incorporated into newly synthesized proteins. The time of centrifugation of the cell lysate used for preparing the cytoplasmic extract was important in obtaining efficient protein synthesis in vitro. When cytoplasmic extracts were prepared from vaccinia‐virus‐infected cells exposed to cordycepin (3′‐deoxyadenosine), an inhibition of 50–70% of the endogenous protein synthesis in vitro was observed, similar to the inhibition seen in vivo. Although the incorporation of formylated methionine from f[35S]Met‐tRNAMetf was not inhibited in the infected cell extracts, the incorporation of [35S]methionine into the N‐terminal position was significantly reduced; since [35S]methionine incorporation (into protein) sensitive to aurintricarboxylic acid and edeine was also reduced in the infected cell extracts, it was concluded that the initiation of polypeptide chains in vitro was inhibited in the vaccinia‐virus‐infected cell extracts. The addition of ribosomal salt wash and spermine to the extracts, although inducing a two–fourfold stimulation of the endogenous protein synthesis in both extracts, did not release the inhibition observed in the infected cell extracts. When the lysates were pretreated with micrococcal nuclease, an inhibition of mRNA‐dependent protein synthesis of approximately 50% was observed in the extracts prepared from vaccinia‐virus‐infected cells exposed to cordycepin. This inhibition occurred either with globin mRNA or with early vaccinia virus mRNA, confirming that the inhibitory factor associated with vaccinia virus did not discriminate between cellular and viral mRNA.