Effects of temperature and lipophilic agents on poliovirus formation and RNA synthesis in a cell-free system

Abstract

The translation and primary processing events of poliovirus polyproteins in HeLa cell extracts were more efficient at 34 degrees C than at 30 or 36 degrees C. The cleavage products of P2 such as 2Apro, 2BC, and 2C appeared early in the reaction before the appearance of the cleavage products of P1 and of 3CDpro, an observation suggesting that P2 was cleaved in cis by 3CDpro. Proteolytic processing of the capsid precursor P1 into VP0, VP1, and VP3 was also more efficient at 34 degrees C than at either 30 or 32 degrees C. Surprisingly, processing of 3CDpro to 3Cpro and 3Dpol was almost completely inhibited at 36 degrees C. The synthesis of virus in the cell extract was greatly enhanced at 34 degrees C over that at 30 or 32 degrees C, whereas incubation at 36 degrees C yielded very little virus. Cerulenin, an inhibitor of lipid synthesis, did not appear to affect virus-specific translation or protein processing, but it almost completely inhibited viral synthesis in vitro. Oleic acid drastically inhibited in vitro translation at 100 microM and in vitro poliovirus synthesis at 25 microM. Addition of HeLa cell smooth membranes partially restored translation but not virus formation. Our observations suggest that in vitro translation, proteolytic processing, and virus formation require intact membranes. Analysis of the in vitro translation products revealed that viral RNA polymerase activity increased linearly during incubation of the translation mixture. RNA polymerase in the crude mixture was inhibited by oleic acid but not by cerulenin. Surprisingly, oleic acid had no direct effect on oligo(U)-primed, poly(A)-dependent poly(U) synthesis catalyzed by purified 3Dpol.