1. Volume 23, Issue 10, Pages 1622-1629 (October 2015)
Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism 
Junfang Deng, Ryan N Ptashkin, Yu Chen, Zhi Cheng, Guangliang Liu, Thien Phan, Xiaoling Deng, Jiehua Zhou, Inhan Lee, Yong Sun Lee, Xiaoyong Bao 
Molecular Therapy 
Volume 23, Issue 10, Pages (October 2015)
DOI: /mt
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
tRF5-GluCTC controls the expression of APOER2. (a) Workflow to identify tRF5-GluCTC-associated mRNAs. (b) Sequence alignment of tRF5-GluCTC with APOER2. The interactive region was identified by both RNAhybrid analysis and the binding assays. (c) The APOER2 mRNAs interacting with biotinylated tRF5-GluCTC was confirmed by RT-PCR, using β-actin as a negative control to demonstrate the binding specificity. (d) The mimic of tRF5-GluCTC decreases APOER2 expression. A549 cells were transfected with 25 nmol/l WT-mimic for tRF5-GluCTC or control mimic (ctrl-mimic). At 15 hours post-transfection, total cell lysate was harvested, followed by western blot to measure the expression of APOER2. (e and f) Antisense against tRF5-GluCTC increases APOER2 expression. A549 cells in a six-well plate were cotransfected with 120 nmol/l antisense oligos, anti-tRF5-GluCTC, or anti-ctrl. After 2 hours post-transfection, cells were mock-infected or infected with respiratory syncytial virus (RSV) at MOI of 1, then harvested at 15 hours p.i. to harvest total RNAs or proteins to measure the APOER2's mRNA by qRT-PCR (e) or APOER2 protein by western blot (f) respectively. FC, fold changes; MOI, multiplicity of infection; p.i., postinfection; qRT-PCR, quantitative real-time polymerase chain reaction.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Targeting elements of tRF5-GluCTC. (a) Construction of the firefly (Pp) luciferase plasmid containing the targeted sequence of APOER2, WT, or mutants to identify the motif(s) critical for tRF5-GluCTC's trans-silencing function. (b) The effect of tRF5-GluCTC on Pp luciferase expression from Pp-WT. A549 cells in a 24-well plate were cotransfected with a Pp-WT and a plasmid expressing renilla Rr luciferase, and 120 nmol/l antisense oligos, anti-tRF5-GluCTC, or anti-ctrl. After 2 hours post-transfection, cells were mock-infected or infected with respiratory syncytial virus (RSV) at MOI of 1. At 15 hours p.i., cells were lysed to measure the luciferase activity. Pp values were first normalized by Rr values yielding relative Pp/Rr values (y-axis). **denotes P value <0.01, relative to the first white bar. (c–e) Targeting motifs. A549 cells, cotransfected with a Pp luciferase plasmid, Pp-WT, Pp-Mut5, Pp-MutM, Pp-Mut3, Pp-Mut3-A, or Pp-Mut3-B (0.1 µg/well of 24-well plate), and a Rr luciferase plasmid were infected with RSV at MOI of 1 (c) or treated with 100 nmol/l WT-mimic oligo (d). Mock infection or the ctrl-mimic was used as a control for c and d respectively. At 15 hours p.i. or 30 hours post-transfection, cells were lysed for luciferase assays. Values at y-axis (Pp/Rr) are a representative of three independent experiments and are expressed as mean ± standard error (SE). * or ** on the bars denotes P value <0.05 or <0.01 respectively, relative to the black bar (Pp-WT-transfected plus RSV-infected or WT-mimic-treated samples). (e) A549 cells in hexaplicate were cotransfected with a Pp-WT plasmid (0.1 µg/well of 24-well plate), an Rr plasmid, and 100 nmol/l mimic (WT or Mut3, please see illustration in a). After 15 hours post-transfection, cells were harvested to measure luciferase activities. Values at y-axis (Pp/Rr) are a representative of three independent experiments and are expressed as mean ± SE. **On the second bar (WT-mimic transfected) denotes P value <0.01, relative to the first bar (ctrl-mimic transfected). MOI, multiplicity of infection; p.i., postinfection.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Antiviral effect of APOER2. (a–c) The effect of APOER2 silencing on RSV replication. A549 cells were transfected with 100 nmol/l siRNA against APOER2 (si-APOER2) or si-ctrl using Lipofectamine At 48 hours post-transfection, cells were mock-infected or infected with RSV at MOI of 1 for 15 hours. (a) Total proteins were prepared to confirm the silencing efficiency of si-APOER2. (b) Infectious particles were measured by immunostaining and the values expressed as pfu/ml. (c) Genome copies were measured by qRT-PCR. (d–f) The effect of APOER2 overexpression on RSV replication. A549 cells in six-well plate were transfected with a plasmid encoding Flag-tagged APOER2 (2 µg/well) or a control plasmid for 30 hours, followed by mock infection or respiratory syncytial virus (RSV) infection (MOI of 1) for 15 hours. (d) Confirmation on Flag-APOER2 expression by western blot. (e) Total infectious particles measurement. (f) Genome copies of RSV. * and ** denotes P < 0.05 and < 0.01 respectively, relative to its ctrl plasmid. MOI, multiplicity of infection; p.i., postinfection; qRT-PCR, quantitative real-time polymerase chain reaction.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
APOER2 interacts with respiratory syncytial virus (RSV) P protein. (a) APOER2 forms a complex with RSV P protein. A549 cells in six-well plate were transfected with 2 µg/well plasmids encoding Flag-tagged APOER2 or it control vectors. At 30 hours post-transfection, cells were mock infected or infected with RSV at MOI of 1. At 6 hours p.i., total cell lysates were immunoprecipitated with an anti-Flag antibody followed by western blot using an anti-RSV antibody to detect associated RSV protein(s). A small aliquot was also prepared before the IP for equal input assays. (b) Overexpressed RSV P protein restores APOER2-inhibited RSV replication. HEK-293 cells at 60–70% confluence were transfected with a plasmid encoding V5-tagged RSV P or N (control protein) or their common control vector. After 30 hours, cells were mock infected or infected with RSV at MOI of 1 for 15 hours. The viral particles in the supernatant were titrated by immune staining and protein overexpression was confirmed by western blot. (c) A mechanism model for tRF5-GluCTC-promoted RSV replication. RSV-induced tRF5-GluCTC targets APOER2, leading to suppressed APOER2 expression and consequently releasing more RSV P protein to facilitate RSV genome replication. IP, immunoprecipitation; MOI, multiplicity of infection; p.i., postinfection; UTR, untranslated region.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions