Optimal flexibility of the linker region of Zika virus NS5 methyltransferase-polymerase is critical for virus replication.

The flavivirus NS5 protein contains an N-terminal methyl-transferase (MTase) connected through a flexible linker with a C-terminal RNA-dependent RNA-polymerase (RdRp) domain, that work cooperatively to replicate and methylate the viral genome. In this study we probed the importance of an evolutionary-conserved hydrophobic residue (Val266) located at the start of the ten-residue interdomain linker of Zika virus (ZIKV) NS5. In flavivirus NS5 crystal structures, the start of the linker forms a 3 helix when NS5 adopts a compact conformation, but becomes disordered or extended in open conformations. Using reverse genetics system, we either introduced rigidity in the linker through mutation to a proline or flexibility through a glycine mutation at position 266. ZIKV NS5 Val 266 to Pro mutation was lethal for viral RNA replication while the Gly mutation was severely attenuated. Serial passaging of cell culture supernatant derived from C6/36 mosquito cells transfected with mutant ZIKV RNA showed that the attenuation can be rescued. Next generation deep sequencing revealed four single nucleotide polymorphisms that occur with an allele frequency >98%. The single non-synonymous NS5 mutation Glu419 to Lys is adjacent to RdRp motif G at the tip of the fingers subdomain, while the remaining three are synonymous variants at nucleotide positions 1403, 4403 and 6653 in the genome. Reverse engineering the changes into the ZIKV NS5/Val266Gly background followed by serial passaging revealed that residue 266 is under strong positive selection to revert back to Val. The interaction of the specific conformation of the NS5 linker with Val at position 266 and the RNA binding motif G region may present a potential strategy for allosteric antiviral drug development.