Regulation of human norovirus VPg nucleotidylylation by ProPol and nucleoside triphosphate binding by its amino terminal sequence in vitro

- ProPol is likely the primary polymerase responsible for hNoV VPg nucleotidylylation.
- Charged amino acids in the amino terminus of VPg regulate its nucleotidylylation.
- First demonstration of direct binding of nucleotide triphosphates by hNoV VPg.
- VPg nucleotidylylation is likely regulated through several mechanisms.

The VPg protein of human Norovirus (hNoV) is a multi-functional protein essential for virus replication. The un-cleaved viral precursor protein, ProPol (NS5-6) was 100-fold more efficient in catalyzing VPg nucleotidylylation than the mature polymerase (Pol, NS6), suggesting a specific intracellular role for ProPol. Sequential and single-point alanine substitutions revealed that several positively charged amino acids in the N-terminal region of VPg regulate its nucleotidylylation by ProPol. We provide evidence that VPg directly binds NTPs, inhibition of binding inhibits nucleotidylylation, and NTP binding appears to involve the first 13 amino acids of the protein. Substitution of multiple positively charged amino acids within the first 12 amino acids of the N-terminal region inhibits nucleotidylylation without affecting binding. Substitution of only Lys20 abolishes nucleotidylylation, but not NTP binding. These studies indicate that positively charged amino acids in the first 20 amino acids of hNoV VPg regulate its nucleotidylylation though several potential mechanisms.