We investigated the roles and biochemical properties of recombinant murine norovirus-1 (MNV-1) 3D in RNA synthesis and virus genome-linked protein (VPg) nucleotidylylation. We therefore expressed VPg and 3D of MNV-1 in . MNV-1 3D exhibited RNA-dependent RNA polymerase (RdRp) activity with poly(A) RNA as a template and MnCl as a cofactor. MNV-1 3D 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 in the presence of MnCl 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 Tyr was the probable target site of guanylylation. Homopolymeric RNAs did not enhance VPg guanylylation, whereas -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 -acting element that stimulates 3D-mediated VPg guanylylation.


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