Fig. 1.
Characterization of the RNA probe used for determining the RNA binding of WhNV protein A in vitro. (A) Schematic of plasmids used for protein AGAA (prot AGAA) and (+)RNA1E expression. RNA1E templates with authentic viral 5′ and 3′ termini of WhNV RNA1 and an inserting EGFP sequence were generated from pAC1E by precisely placing the Ac5 promoter start site and a hepatitisδribozyme (Rz), respectively, and by mutating the start codon at the indicated location to disrupt translation. The Ac5 promoter and SV40 polyadenylation signal (SV) flanking the protein A ORF in pAGAA thereof disrupt its activity as a viral RNA replication template and mutating the replication GDD sites into GAA but maintain its activity to recruit RNA (Qiu et al., 2014). pAGAA-derived protein AGAA subsequently directs (+)RNA1E recruitment from (+)RNA1E template transcribed from pAC1E. (B) The secondary structure predicted for RNA1 nt 50–118 [RNA1(50–118)]. Del represents removing the RNA sequences formed the helices structure and Mut represents destroying the base pairing in the helices section. (C) RNA1(50–118) mediates RNA1 recruitment in cells. Pr-E cells were transfected with the indicated plasmids, including pAC1E wt, Del or Mut (as shown in B) in the absence or in the presence of pAGAA (protein AGAA). After transfection for 36 h, total RNA was extracted and analyzed by Northern blot with the probes against EGFP and 18S rRNA, respectively. (D) The levels of (+)RNA1E were determined from three experiments after normalization to 18S rRNA and are expressed as the level of protein A-stimulated (+)RNA1E accumulation relative to wt (+)RNA1E. (E) The secondary structure predicted for RNA2 nt 123–164 [RNA1(123–164)]. Del′ represents removing the RNA sequences formed the helices structure. (F) Schematic of plasmids used for protein A-mediated RNA2 recruitment. (G) RNA2(123–164) mediates RNA2 recruitment in cells. Pr-E cells were transfected with the indicated plasmids, including pAC2 wt, pAC2 181–1562, pAC2 1–180, pAC2 Del′ (as shown in E and F) in the absence or in the presence of pAGAA. After transfection for 36 h, total RNA was extracted and analyzed by Northern blot with the probes against EGFP and 18S rRNA, respectively. (H) The levels of (+)RNA2 were determined from three experiments after normalization to 18S rRNA and are expressed as the level of protein A-stimulated (+)RNA2 accumulation relative to wt (+)RNA2.