Conversion of VPg into VPgpUpUOH before and during poliovirus negative-strand RNA synthesis

J Virol. 2009 Dec;83(24):12660-70. doi: 10.1128/JVI.01676-08. Epub 2009 Oct 7.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Carrier Proteins / physiology
  • Deoxycytosine Nucleotides / metabolism*
  • Guanidine / pharmacology
  • HeLa Cells
  • Humans
  • Poliovirus / genetics*
  • Poliovirus / metabolism
  • RNA, Viral / biosynthesis*
  • Regulatory Sequences, Nucleic Acid
  • Ribonuclease T1
  • Uridine / metabolism*
  • Viral Nonstructural Proteins / physiology
  • Viral Proteins / metabolism*

Substances

  • Carrier Proteins
  • Deoxycytosine Nucleotides
  • RNA, Viral
  • VPg protein, poliovirus
  • Viral Nonstructural Proteins
  • Viral Proteins
  • 2'-deoxycytidine 5'-triphosphate
  • Ribonuclease T1
  • 2C protein, viral
  • Guanidine
  • Uridine