(A) Total viruses were harvested and infectious particles were quantified by immunostaining using an anti-RSV antibody. development against RSV infection. 0.01, relative to the white bar. (E) Left panel: Sequence alignment of tRF5-GlnCTG with a sensor plasmid containing targeting sequence of tRF5-GlnCTG (luciferase sensor plasmids and internal control renilla luciferase plasmids (values) was calculated. Data are representative of three independent experiments. * 0.05, relative to the paired white bar. Quantitative real-time PCR (qRT-PCR) qRT-PCR quantification of tRF5-GlnCTG was developed as described with some modifications (Zhou et al., 2017b). In brief, extracted cellular RNA was treated with T4 polynucleotide kinase (T4PNK) to convert the 3 cyclic phosphate group into a hydroxyl group and ligated to a Xanthiside 3 RNA linker using T4 RNA ligase, followed by cDNA synthesis. A forward primer specific to tRF5-GlnCTG or Enpep RNU6 and a reverse primer specific to a 3 linker were used to amplify targets. qRT-PCR was performed by using iTaq? Universal SYBR Green Supermix (Bio-rad) in the CFX Connect Real-Time PCR System (Bio-rad) under the following conditions: initial steps: 50C, 2?min, and 95C, 10?min; PCR steps: 95C, 15 sec and 60 C, 1?min for 40 cycles. The sequences of primers and a 3 RNA linker are shown in Figure 2A and Supplementary Table S1. Construction of luciferase sensor plasmids and luciferase assays To identify the functional domain(s) of tRF5-GlnCTG, sensor plasmids containing a complementary sequence of wild-type tRF5-GlnCTG (values were normalized by renilla luciferase (for 6?h, followed by mock- or RSV infection. At 24 h post-infection (p.i.), cells were lysed for a luciferase assay using a dual-luciferase kit (Promega, Madison, WI). The relative luciferase activity values) was calculated and compared between WT and mutants. To investigate the effect of RSV proteins on tRF5-GlnCTG induction, A549 cells were transfected with a plasmid encoding V5-tagged RSV N, P, NS1, or NS2 protein in the presence of a expression vectors were also co-transfected. At 30?h post-transfection, cells were lysed and followed by luciferase measurement. A plasmid expressing V5 only was used as a negative control for viral protein expression. RNA interference 100?nmol/l of siRNA against ANG, RNase L, Dicer, Drosha, or ELAC2 was transfected Xanthiside into A549 cells using Lipofectamine 2000 (Invitrogen). After Xanthiside 40?h, A549 cells were mock- or RSV-infected for 24 h at an MOI of 1 1, followed by Northern blot (NB), qRT-PCR, or Western blot. The sequence of siRNAs and primers for qRT-PCR is shown in Supplementary Table S1. Western blot (WB) The cell lysates, after protein quantification, were fractionated in SDS-PAGE denaturing gels, as previously described (Choi et al., 2018). The separated proteins were then transferred to polyvinylidene difluoride membranes, followed by incubation with the proper primary antibodies according to the manufacturers instructions. Subcellular fractionation Two types of fractionation were performed. One was to isolate nuclei from the total cells, resulting in nuclear and mitochondria-containing cytoplasmic samples, similarly as we described before (Wang et al., 2013). Another one was to prepare the nuclear, mitochondria-free cytoplasmic, and mitochondrial fractions, using the Qproteome Mitochondria Isolation Kit from Qiagen (37612; Valencia, CA), according to manufacturer’s instructions. Briefly, A549 cells, mock- or RSV-infected, were washed with 0.9% sodium chloride solution and resuspended in ice-cold lysis buffer. After incubation on ice for 10?min, cell lysates were centrifuged at 1000 x g for 10?min at 4C and the supernatants were stored as mitochondria-free cytoplasmic fraction. The pellet was resuspended in ice-cold Disruption Buffer and completely disrupted using a syringe with a blunt-ended needle, followed by centrifugation at 1000 x g for 10?min at 4C. The pellet was stored as the nuclear fraction. The supernatant was further centrifuged at 6000 x g for 10? min at 4C and the mitochondrial pellet was more purified in the layer of Mitochondria purification and Disruption Buffer. RNA and protein samples of the nuclear, mitochondria-free cytoplasmic, and mitochondrial fractions were prepared for NB and WB, respectively. Immunoprecipitation 293 cells.
