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/helicase 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 containing 2 mM guanidine HCI, a reversible inhibitor of viral protein 2C. Upon incubation in reactions lacking guanidine, preinitiation RNA replication complexes synchronously initiated and elongated negative-strand RNA molecules, followed by the synchronous initiation and elongation of positive-strand RNA molecules. The immediate and exclusive synthesis of negative-strand RNA upon the removal of guanidine demonstrates that guanidine specifically blocks the initiation of negative-strand RNA synthesis. Readdition of guanidine HCl to reactions synchronously elongating nascent negative-strand RNA molecules did not prevent their continued elongation and completion. In fact, readdition of guanidine HCl to reactions containing preinitiation complexes elongating nascent negative-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 negative-strand RNA molecules, the initiation of positive-strand RNA molecules, or the elongation of positive-strand RNA molecules. The guanidine-inhibited function of viral protein 2C is required only immediately before or during the initiation of negative-strand RNA synthesis. We suggest that guanidine may block an irreversible structural maturation of protein 2C and/or RNA replication complexes necessary for the initiation of RNA replication.
Barton
D J
DJ
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville 32610, USA.
Flanegan
J B
JB
D002352:Carrier Proteins / Q000502:physiology*; D019791:Guanidine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D012367:RNA, Viral / Q000096:biosynthesis*; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication / Q000187:drug effects
D002352:Carrier Proteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C076023:2C protein, viral; D019791:Guanidine