Effects of RNA polymerase inhibitors on nucleotidylylation of human norovirus VPg

Replication of human norovirus requires nucleotidylylation of VPg by a viral polymerase, either mature Pol or the precursor protein ProPol. The reaction catalyses the covalent attachment of a nucleoside monophosphate to tyrosine 27 of HuNV VPg, which forms the first nucleotide of the genome and primes RNA replication. VPg at the genomic 5' terminus is also critical for translation initiation. Despite the importance of nucleotidylylation, the effect of antivirals on the reaction is not well characterized. This work investigated the effect of norovirus polymerase inhibitors on nucleotidylylation of VPg. A gel-based assay was developed whereby nucleotidylylation of GII.4 HuNV VPg was visualized by SDS-PAGE and the linkage of nucleotide(s) confirmed by mass spectrometry. Non-nucleotide inhibitors decreased nucleotidylylation of VPg in a dose-dependent manner, with PPNDS and NF023 being the most effective. Nucleotidylylation in the presence of the nucleotide analogues ddhGTP, ddhUTP or 2CMC-TP had no effect on the reaction nor were the nucleotides substrates for the linkage to VPg. Tenofovir diphosphate, while not utilised as a substrate, did cause a dose-dependent decrease in CTP-mediated nucleotidylylation. Favipiravir-TP and 5F-UTP, analogues with modified nitrogenous Bases, were poorly utilised as substrates for nucleotidylylation and showed 56% and 58% labelling of VPg respectively at 800 μM. Finally, a malachite green assay to measure release of PPi was developed as a method for high-throughput screening of antivirals that target nucleotidylylation. This data demonstrates that nucleotidylylation of VPg is a target for Antiviral screening and that different classes of antivirals have variable effects on nucleotidylylation.