Conversion of VPg into VPgpUpUOH before and during poliovirus negative-strand RNA synthesis.
There are two protein primers involved in picornavirus RNA replication, VPg, the viral protein of the genome, and VPgpUpU(OH). A cis-acting replication element (CRE) within the open reading frame of poliovirus (PV) RNA allows the viral RNA-dependent RNA polymerase 3D(Pol) to catalyze the conversion of VPg into VPgpUpU(OH). In this study, we used preinitiation RNA replication complexes (PIRCs) to determine when CRE-dependent VPg uridylylation occurs relative to the sequential synthesis of negative- and positive-strand RNA. Guanidine HCl (2 mM), a reversible inhibitor of PV 2C(ATPase), prevented CRE-dependent VPgpUpU(OH) synthesis and the initiation of negative-strand RNA synthesis. VPgpUpU(OH) and nascent negative-strand RNA molecules were synthesized coincident in time following the removal of guanidine, consistent with PV RNA functioning simultaneously as a template for CRE-dependent VPgpUpU(OH) synthesis and negative-strand RNA synthesis. The amounts of [(32)P]UMP incorporated into VPgpUpU(OH) and negative-strand RNA products indicated that 100 to 400 VPgpUpU(OH) molecules were made coincident in time with each negative-strand RNA. 3'-dCTP inhibited the elongation of nascent negative-strand RNAs without affecting CRE-dependent VPg uridylylation. A 3' nontranslated region mutation which inhibited negative-strand RNA synthesis did not inhibit CRE-dependent VPg uridylylation. Together, the data implicate 2C(ATPase) in the mechanisms whereby PV RNA functions as a template for reiterative CRE-dependent VPg uridylylation before and during negative-strand RNA synthesis.
Steil
Benjamin P
BP
University of Colorado Denver, School of Medicine, Department of Microbiology, MS8333, P.O. Box 6511, 12800 East 19th Ave., Aurora, CO 80045, USA.
Barton
David J
DJ
D002352:Carrier Proteins / Q000502:physiology; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D017955:Poliovirus / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012045:Regulatory Sequences, Nucleic Acid; D006163:Ribonuclease T1; D014529:Uridine / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000502:physiology; D014764:Viral Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D003845:Deoxycytosine Nucleotides; D012367:RNA, Viral; C520979:VPg protein, poliovirus; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C024107:2'-deoxycytidine 5'-triphosphate; D006163:Ribonuclease T1; C076023:2C protein, viral; D019791:Guanidine; D014529:Uridine