Superior RdRp Function Drives the Dominance of Prevalent GI.3 Norovirus Lineages

The GI.3 norovirus is the most detected and recombinant-rich genotype within genogroup I, yet the mechanistic basis for its epidemiological success remains poorly understood. This study integrates Bayesian evolutionary analysis with in vitro enzymology to investigate the link between RdRp function and the evolutionary dynamics of GI.3 NoV. We analyzed 831 GI.3 sequences, finding that prevalent strains (GI.3[P3] and GI.3[P13]) exhibited significantly higher evolutionary rates in both the RdRp and VP1 genes than non-prevalent strains (GI.3[P10] and GI.3[P14]). While the RdRp gene displayed a strong molecular clock signal, the VP1 gene's evolution was more complex, showing cluster-specific trends. Functionally, the RdRps from prevalent strains demonstrated superior enzymatic activity and substrate affinity (K_m: GI.3[P13] = 0.092 mM; GI.3[P3] = 0.176 mM) compared to non-prevalent strains (K_m: GI.3[P14] = 0.273 mM). Notably, GI.3 RdRp required higher manganese ion concentrations for optimal activity than previously reported for GII strains, suggesting a potential biochemical constraint. Our findings demonstrate a clear correlation between RdRp enzymatic efficiency, evolutionary rate, and strain prevalence. We propose that a highly active RdRp may potentially accelerate VP1 evolution and confer a replicative advantage, underpinning the dominance of specific GI.3 lineages. This work provides crucial experimental evidence linking viral polymerase function to evolutionary and epidemiological outcomes.