Key result
Recombinant MNV-1 3Dpol exhibited RNA-dependent RNA polymerase activity and guanylylated VPg at Tyr117, which was enhanced by the (-)ORF3 region of negative-strand RNA.
MNV-1 3Dpol demonstrates RNA-dependent RNA polymerase activity and guanylylates VPg at Tyr117, providing insight into norovirus replication mechanisms.
Should not yet alter clinical practice; leaves open norovirus polymerase as antiviral target pending human validation.
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 MnCl(2) as a cofactor. MNV-1 3D(pol) demonstrated optimum RNA-synthesis activity at pH 7.4 and 37 degrees C in the absence of a primer. Further, VPg was guanylylated by MNV-1 3D(pol) in the presence of MnCl(2) in a template-independent manner. The guanylylation reaction conducted with VPg substitution mutants (Y26F, Y40F, Y45F and Y117F) and a deletion mutant (Delta117-124) indicated that Tyr(117) 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.
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Han et al. (2010) studied Murine norovirus-1 (in vitro). Recombinant murine norovirus-1 (MNV-1) 3Dpol and VPg was evaluated on RNA-dependent RNA polymerase (RdRp) activity and VPg nucleotidylylation. Recombinant MNV-1 3Dpol exhibited RNA-dependent RNA polymerase activity and guanylylated VPg at Tyr117, which was enhanced by the (-)ORF3 region of negative-strand RNA.
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