Journal: PLoS Pathogens
Article Title: Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication
Figure Lengend Snippet: Identification of RyDEN. (A) Procedure for gain-of-function screen. The cDNA library was generated from mRNA of IFN-α/ω-treated HeLa cells and transferred into a lentiviral vector by the Gateway recombination system. Infectious lentiviral vectors carrying the IFN cDNA library were produced as a VSV-G-pseudotyped virus and used to transduce DENV-susceptible Huh7.5 cells. cDNA library-expressing Huh7.5 cells were then challenged with DENV-2 at an MOI of 1, and cell colonies that survived DENV-induced cell death were collected. (B) Histogram analysis of cDNA fragments in library vectors. The entry vector (pDONR221, left panel) and destination vector (pYK005C, right panel) recombinated with the Gateway-compatible cDNA library were applied to Escherichia coli ( E . coli) , and the cells were spread onto LB plates to develop bacterial colonies. The cDNA fragments in individual colonies were amplified by PCR using primers described in Materials and Methods and visualized with agarose gel electrophoresis. The size of the PCR fragment was estimated by comparing the migration distance of the DNA molecular weight markers. Up to sixty colonies were picked up from each vector-transformed E . coli plate and analyzed. (C) Validation of DENV-resistant cell clones. Surviving clones obtained from (A) were seeded in a chamber slide and infected with DENV-2 at an MOI of 5. Two days after infection, cells were fixed with paraformaldehyde, permeabilized, and stained with anti-dsRNA antibody, followed by detection with Alexa Fluor 488-conjugated secondary antibody (red). Cell nuclei were stained with DAPI (blue). Representative merged images using four surviving clones (#1, 13, 14, and 15) and control cells (bacterial alkaline phosphatase [BAP]-expressing Huh7.5 cells) are shown. In a parallel experiment, the culture supernatant of infected cells was harvested 2 days after infection and subjected to plaque assay to measure the virus titer (insets). (D) Amplification of cDNA from DENV-resistant cells. Genomic DNA was isolated from cell clones, whose resistant property had been confirmed in (B), and cDNA was amplified by PCR using primers specific to the lentiviral vector. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. (E) Amino acid sequence of RyDEN. (F) Predicted domain organization of RyDEN. RyDEN protein (291 amino acid) was suggested to contain eight α-helixes (blue), seven β-strands (orange), NLS (121–137), NES (261–269), zinc-ribbon domain (112–135), and coiled-coil motif (261–285). A unique glutamic acid-rich (E-rich) domain was also found in the C-terminus.
Article Snippet: Generation of lentiviral vectors carrying an IFN cDNA library A Gateway-compatible cDNA library was generated from mRNA isolated from HeLa cells that had been treated with 1,000 U/ml type I IFN (a mixture of human interferon α and ω, Sigma) for 24 h. Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen), and mRNA was then isolated using a PolyATtract mRNA Isolation System II (Promega) according to the manufacturer’s recommendations.
Techniques: cDNA Library Assay, Generated, Plasmid Preparation, Produced, Transduction, Expressing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Migration, Molecular Weight, Transformation Assay, Clone Assay, Infection, Staining, Plaque Assay, Isolation, Sequencing