Fig 1: A highly conserved region of the MTase C-terminus is required for PAF1C binding.(A) The protein interaction between flavivirus NS5s and PAF1 was tested biochemically. Plasmids encoding NS5s and GFP were transfected and affinity purified in HEK293T cells via a 2xStrep II tag. Immunoblot analysis of lysate and purified (Strep-AP) fractions were performed against PAF1, Strep, and GAPDH (loading/negative control). (B) Logo analysis of amino acid conservation for flavivirus NS5s from (A) in the PAF1-interacting region. The conserved stretch from amino acids 258–263 (magenta) was targeted for alanine scanning leading to the generation of 2 NS5 mutants: LGS258AAA (NS5LGS) and GTR261AAA (NS5GTR). (C) DENV2 NS5 structure from PDB (5ZQK) with PAF1-interacting region highlighted (box). The PAF1-interacting region overlaps with the C-terminal end of the MTase (yellow) and the flexible linker domain (cyan), but not the RdRP (grey). The PAF1-interacting region includes a stretch of amino acids conserved in PAF1-interacting flaviviruses tested in (A). (D) Mutants from (B) were tested for an interaction with PAF1C biochemically. Purification and immunoblot analysis were conducted as in (A). PAF1C complex members CTR9, LEO1, CDC73 and PAF1 were probed. STAT2 was used as a positive control for an NS5 interaction outside of the PAF1C-interacting region. Abbreviations: Zika virus (ZIKV), West Nile virus (WNV), Powassan virus (POWV), Langat virus (LGTV), tick-borne encephalitis virus (TBEV), Strep-affinity purified (Strep-AP).