Rna-Dependent RNA polymerase of japanese encephalitis virus binds the initiator nucleotide gtp to form a mechanistically important pre-Initiation state.
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.
