VIROLOGY 161,366-373 (1987) Selective inhibition of Protein Synthesis by Synthetic and Vaccinia Virus-Core Synthesized Poly(riboadenylic acids) ROSTOM BABlANIAN,**’ SHYAMAL K. GOSWAMI,” MARIANO ESTEBAN,* AND AMIYA K. BANERJEEt *Department of Microbiology and Immunology, SUNY, Health Sciences Center at Brooklyn, Brooklyn, New York 11203, and tRoche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 Received April 1 I 1987; accepted July 25, 1987 Studies were undertaken to compare the effect of poly(A)s from various sources on selective inhibition of protein synthesis in the reticulocyte lysate system programmed with viral and cellular mRNAs. RNA synthesized in vitro by vaccinia virus cores in the presence of only ATP inhibited overall HeLa cell polypeptide synthesis by over 80% with a minimal effect on translation of vaccinia virus mRNAs. Hybridization of the [cu-32P]AMP-labeled RNA made in vitro by vaccinia virus cores in the presence of only ATP, showed no complementarity to Hindlll restriction fragments of vaccinia virus DNA indicating that the in vitro product was poly(A). Fractionation of synthetic and core-synthesized poly(A) into three size classes showed that the larger the size of poly(A), the greater its inhibitory activity of protein synthesis in the cell-free system. Inhibition of translation of mRNAs from vaccinia virus-infected HeLa cells was also observed in the presence of poly(A). However, virus-induced polypeptide synthesis was more resistant to the effect of poly(A) than were cellular polypeptides. Oligo(dT) when added to the reticulocyte lysate system was capable of reversing the inhibition of protein synthesis caused by both core-synthesized poly(A) and core-transcribed RNAs. These results indicate that poly(A) synthesized by the virion-associated enzyme has inhibitory properties similar to those of synthetic poly(A). 0 1997 Academic Press. Inc. INTRODUCTION Vaccinia virus infection causes a striking inhibition of host cell protein synthesis (Kit and Dubbs, 1962; Shatkin, 1963; Salzman and Sebring, 1967; Holowc- zak and Joklik, 1967; Moss and Salzman, 1968; Metz and Esteban, 1972). This rapid inhibition of host cell protein synthesis with the concurrent synthesis of vaccinia virus polypeptides is a selective inhibition; also known as shutoff. During this selective inhibition, host cell polysomes are degraded (Joklik and Merigan, 1966) and viral polysomes are formed (Metz et a/., 1975). The mechanism of this selective inhibition has been extensively studied and several hypotheses have been proposed for the shutoff phenomenon (for review see Bablanian, 1984). Some of these studies have demonstrated a relationship between vaccinia virus- induced RNA synthesis and the inhibition of host cell protein synthesis (Bablanian et al., 1981a, b). Other studies have suggested that vaccinia virus-directed small RNA molecules, produced either in vivo or in vitro, may play a role in shut-off (Rosmond-Hornbeak and Moss, 1975; Gershowitz and Moss, 1979; Paoletti et a/., 1980). Our laboratory has previously demon- strated (Coppola and Bablanian, 1983) that in vitro transcribed vaccinia virus RNA inhibited translation of ’ To whom requests for reprints should be addressed. several host and viral mRNAs in the reticulocyte lysate system, but minimally affected the translation of vac- cinia virus mRNAs. Further characterization of the core-synthesized RNAs revealed that the small poly(A)-containing fraction of this RNA differentially in- hibited translation of HeLa cell mRNA and of vaccinia virus mRNA. On the other hand, the larger poly(A)- containing RNA fractions from the in vitro transcription which translated efficiently in the reticulocyte system, no longer possessed the selective inhibitory activity; instead, a direct translational competition was seen between HeLa and vaccinia virus mRNAs when they were added together in the reticulocyte lysate system (Bablanian et al., 1986). A series of experiments was under-taken in order to determine what type of struc- tural changes of the small in vitro vaccinia virus tran- scripts are necessary to abrogate this selective inhibi- tory activity. It was established that the decapping of in vitro viral transcripts by @-elimination, using unmethy- lated transcripts, and finally using IMP-containing transcripts to cause relaxation of the secondary struc- ture of RNA, all had no effect on the selective inhibitory property of the in vitro transcripts (R. Bablanian and A. K. Banerjee, unpublished results). The only proce- dure which eliminated this selective inhibitory activity of the in vitro transcribed RNAs was treatment of the transcripts with ribonuclease H and oligo(dT) (Bablan- ian and Banerjee, 1986). Since this treatment removes 0042-6822187 $3.00 Copyright 8 1997 by Academic Press. Inc. All rights of repmduction in any form resewed. 366