Fig. 2
Bioengineered r/si-ORF7 was processed to mature si-ORF7 and reduced virus copy numbers. A A genome map for three different VZV strains. TRL, terminal repeat long; IRL, internal repeat long; UL, unique long, IRs, terminal repeat short; Us, unique short. A highly conserved region of the VZV ORF7 was target by si-ORF7. The target site and sequence for si-ORF7 recognition on ORF7 gene was shown below the map (si-ORF7 sequence is shown in red and the viral sequence in black). B Schematic illustration of the novel hybrid tRNASer scaffold used to produce r/si-ORF7. The location of mature si-ORF7 sequence was shown in red. C Urea-PAGE analysis of total bacterial RNAs showed that chimeric r/si-ORF7 was heterogeneously expressed in E. coli using a novel hybrid tRNASer scaffold. Total RNAs isolated from untransformed HST08 (blank) E. coli were used as control. r/si-ORF7 and SSA were expressed at much higher levels marked by red arrow. D Urea-PAGE analyses of the collected fractions eluted at 24.5 min, which confirmed the purity of isolated r/si-ORF7. E–G Virus copy numbers were detected by dPCR. ARPE-19 cells were transfected with 10 nM of r/si-ORF7 6 h before infection by VZV v-Oka at moi of 0.3 and incubated for 2 days. Positive droplets were showed in both r/si-ORF7 (E) and SSA (F) 2 days post-infection by QutantaSofts’ddPCR fluorescence readouts. G the copy numbers of VZV vOka (ORF68 was selected to detect). Values are the mean ± S.D. of triplicated treatments; ***P < 0.001 relative to SSA. H–K The process of r/si-ORF7 to mature si-ORF7. ARPE-19 cells were transfected with 10 nM of r/si-ORF7 6 h before infection by VZV v-Oka at moi of 0.3 and incubated for 2 days. The level of tRNA scaffold (H), precursor of mature si-ORF7 (I), mature si-ORF7 (J), and ORF7 mRNA expression level (K) were detected 48 h post-transfection. Values are the mean ± S.D. of triplicated treatments; ***P < 0.001 vs. SSA. I ARPE-19 cells were transfected with 10 nM of r/si-ORF7. Mature si-ORF7 expression was detected by stem loop-RT qPCR at 1, 2, 3, and 4 days post-transfection. Values are the mean ± S.D. of triplicated treatments; ***P < 0.001 vs. SSA. M ARPE-19 cells were transfected with 10 nM to 100 nM r/si-ORF7. CCK8 assay was undertaken to measure the cell viability of r/si-ORF7 on ARPE-19 cells