Mutation of a conserved lysine in the RdRp fingers domain broadly attenuates orthobunyaviruses.

Orthobunyaviruses, comprising more than 130 species, constitute a significant threat to human and animal health, yet no effective broad-spectrum treatments or vaccines are currently available. Here, we initially identified an evolutionarily constrained lysine residue, highly conserved across all 22 Orthobunyavirus serogroups, within the α30 helix of the RNA-dependent RNA polymerase (RdRp). Functional interrogation of this site by charge-reversal substitution (K-to-E) substantially impaired polymerase activity and led to marked attenuation of Ebinur Lake virus (EBIV), as evidenced by impaired replication, attenuated cytopathogenicity, and reduced virulence in mice. The similar attenuation observed in Bunyamwera virus (BUNV), the prototype virus of the genus, and Oya virus (OYAV), a member of the Simbu serogroup, demonstrates that this residue fulfills a conserved functional role. Biochemical and cellular analyses further revealed a graded, charge-dependent mutational effect, indicating that attenuation arises predominantly from disruption of the essential interaction between RdRp and the 3' terminus of viral genomic RNA (3' vRNA). Structural modeling and mutagenesis further suggest that the K-to-E substitution creates a new hydrogen bond that may also contribute to viral attenuation. Together, these findings establish the conserved lysine as a critical regulator of RdRp function and offer a rational strategy for broad-spectrum attenuation of orthobunyaviruses.