Nothing Special   »   [go: up one dir, main page]

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Nucleic Acids Res. 2014 Feb;42(4):2758-73. doi: 10.1093/nar/gkt1106. Epub 2013 Nov 28.

Abstract

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRpGTP structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRpGTP structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn(2+) ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a checkpoint to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / chemistry
  • Amino Acid Motifs
  • Binding Sites
  • Encephalitis Virus, Japanese / enzymology*
  • Guanosine Triphosphate / chemistry*
  • Guanosine Triphosphate / metabolism
  • Models, Molecular
  • RNA / biosynthesis*
  • RNA / metabolism
  • RNA-Dependent RNA Polymerase / chemistry*
  • RNA-Dependent RNA Polymerase / metabolism

Substances

  • RNA
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • RNA-Dependent RNA Polymerase

Associated data

  • PDB/4HDG
  • PDB/4HDH
  • PDB/4MTP