Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg

We investigated the roles and biochemical properties of recombinant murine norovirus-1 (MNV-1) 3D^pol in RNA synthesis and virus genome-linked protein (VPg) nucleotidylylation. We therefore expressed VPg and 3D^pol of MNV-1 in Escherichia coli. MNV-1 3D^pol exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro with poly(A) RNA as a template and MnCl₂ as a cofactor. MNV-1 3D^pol demonstrated optimum RNA-synthesis activity at pH 7.4 and 37 °C in the absence of a primer. Further, VPg was guanylylated by MNV-1 3D^pol in the presence of MnCl₂ in a template-independent manner. The guanylylation reaction conducted with VPg substitution mutants (Y26F, Y40F, Y45F and Y117F) and a deletion mutant (Δ117–124) indicated that Tyr¹¹⁷ was the probable target site of guanylylation. Homopolymeric RNAs did not enhance VPg guanylylation, whereas in vitro-transcribed (−) subgenomic (SG) and (+)SG RNA enhanced VPg guanylylation by 9.2 and 3.2 times, respectively. Within (−)SG RNA, the (−)ORF3 region played a critical role in enhancing VPg guanylylation, suggesting that the MNV-1 ORF3 region of negative-strand RNA contains a cis-acting element that stimulates 3D^pol-mediated VPg guanylylation.