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UGGT1 enhances viral RNA replication.

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posted on 2017-05-17, 17:55 authored by Peng-Nien Huang, Jia-Rong Jheng, Jamie J. Arnold, Jen-Ren Wang, Craig E. Cameron, Shin-Ru Shih

(A) NC or UGGT1 siRNA-treated RD cells were transfected with EVA71-Luc replicon RNA, and cells were assayed for firefly luciferase signals (FLuc) at 6 h post-transfection. The right panel indicates the knockdown efficiency of Uggt1. (B) Monocistronic mRNA containing EVA71 IRES and FLuc was transfected to cells pretreated with NC or UGGT1 siRNA. At 6 h post-transfection, cell lysates were assayed for FLuc activity. Western blotting data indicates siRNA knockdown efficiency. Experiments were performed in triplicate to obtain the bar graph. (C) NC or UGGT1 siRNA-treated RD cells were infected with EVA71 at an MOI of 10. Intracellular viral RNA was isolated at 4, 6, 8, 10, 12, and 14 h post-infection, and quantitated using real-time RT-PCR. The amount of viral RNA at 14 h post-infection in NC siRNA-transfected cells was taken as 100%, and the relative amount of viral RNA isolated at each timepoint is presented as a percentage of this. The right panel indicates knockdown efficiency of Uggt1. (D) RD cells were transfected with NC or UGGT1 siRNA for 48 h and then reseeded. After 24 h, cells were infected with EVA71 at an MOI of 10, and RNA was extracted at 2, 4, 6, 8, and 10 h post-infection. RNA was loaded onto a nitrocellulose sheet in the slot blot manifold. The right panel demonstrates Uggt1 knockdown efficiency. ***P < 0.001 and *P < 0.05, as calculated by two-tailed unpaired Student’s t-test.