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. 2019 Sep 5;116(38):19136–19144. doi: 10.1073/pnas.1905617116

Fig. 4.

Fig. 4.

Ae. aegypti ME31B binds to the second dumbbell RNA structure of ZIKV and WNV sfRNA. (A, Top) Schematic overview of the used plasmid constructs. The genes of interest (GOI) were expressed fused N-terminally to EGFP followed by a foot-and-mouth disease virus 2A ribosome skipping sequence and a triple FLAG (3F)-tag which allows the expression of separate EGFP-2A and 3F-GOI. (A, Bottom) RNA-affinity purification to confirm ZIKV and WNV sfRNA binding proteins. Lysates of Aag2 cells transfected with pPUB-3F-ME31B, pPUB-3F-ATX2, pPUB-3F-LSM12, or pPUB-3F-60sRp were prepared. Lysates were subjected to RNA-affinity purification using streptavidin beads coated with in vitro transcribed RNA of 4XS1m-ZIKV-sfRNA, 4XS1m-WNV-sfRNA, or 4XS1m-control. RNA-bound proteins were eluted with RNase A and detected by Western blot with α-Flag antibodies. (B) Lysates of Aag2 cells transfected with pPUB-3F-ME31B were subjected to RNA-affinity purification with 4XS1m-aptamer fused 3′-truncated sfRNAs of ZIKV and WNV. A schematic overview of the used truncations is included in the figure. RNA-bound proteins were eluted with RNase A and detected by Western blot with α-Flag antibodies.