COMPLETE REPLICATION OF POLIOVIRUS IN-VITRO - PREINITIATION RNA REPLICATION COMPLEXES REQUIRE SOLUBLE CELLULAR FACTORS FOR THE SYNTHESIS OFVPG-LINKED RNA

Translation of poliovirion RNA in HeLa S10 extracts resulted in the fo rmation of RNA replication complexes which catalyzed the asymmetric re plication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replicatio n complexes isolated from HeLa S10 translation reactions by pulse-labe ling with [P-32]CTP. The RNA replication complexes formed in vitro con tained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Geno me-length poliovirus RNA covalently linked to VPg was synthesized in l arge amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both an ti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting vira l protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes i n reaction mixes containing 2 mM guanidine HCl. Preinitiation complexe s sedimented upon centrifugation at 15,000 x g and initiated RNA repli cation upon their resuspension in reaction mixes lacking guanidine. In itiation of RNA synthesis by preinitiation complexes did not require a ctive protein synthesis pr the addition of soluble viral proteins. Ini tiation of RNA synthesis by preinitiation complexes, however, was abso lutely dependent on soluble HeLa cytoplasmic factors. Preinitiation co mplexes also catalyzed the formation of infectious virus in reaction m ixes containing exogenously added capsid proteins. The titer of infect ious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions repre sent an efficient in vitro system for authentic poliovirus replication , including protein synthesis, polyprotein processing, RNA replication , and virus assembly.