SYNCHRONOUS REPLICATION OF POLIOVIRUS RNA - INITIATION OF NEGATIVE-STRAND RNA-SYNTHESIS REQUIRES THE GUANIDINE-INHIBITED ACTIVITY OF PROTEIN 2C

We report that protein 2C, the putative nucleoside triphosphatase/heli case protein of poliovirus, is required for the initiation of negative -strand RNA synthesis. Preinitiation RNA replication complexes formed upon the translation of poliovirion RNA in HeLa S10 extracts containin g 2 mM guanidine HCl, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replicat ion complexes synchronously initiated and elongated negative-strand RN A molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis o f negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA sy nthesis. Readdition of guanidine HCl to reactions synchronously elonga ting nascent negative-strand RNA molecules did not prevent their conti nued elongation and completion. In fact, readdition of guanidine HCl t o reactions containing preinitiation complexes elongating nascent nega tive-strand RNA molecules had no effect on subsequent positive-strand RNA synthesis initiation or elongation. Thus, the guanidine-inhibited function of viral protein 2C was not required for the elongation of ne gative-strand RNA molecules, the initiation of positive-strand RNA mol ecules, or the elongation of positive-strand RNA molecules. The guanid ine-inhibited function of viral protein 2C is required only immediatel y before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturatio n of protein 2C and/or RNA replication complexes necessary for the ini tiation of RNA replication.