Topoisomerase III-ß is required for efficient replication of positive-sense RNA viruses

K. Reddisiva Prasanth; Minato Hirano; W. Samuel Fagg; Eileen T. McAnarney; Chao Shan; Xuping Xie; Adam Hage; Colette A. Pietzsch; Alexander Bukreyev; Ricardo Rajsbaum; Pei-Yong Shi; Mark T. Bedford; Shelton S. Bradrick; Vineet Menachery; Mariano A. Garcia-Blanco

TDRD3 KO cells, HuH-7 cells were co-transfected with pX330-TDRD3 gRNA1, pX330-TDRD3 gRNA2, and puromycin plasmid. At the second day of culture, puromycin (2mg/ml) was supplemented into the culture medium, and the cells with recombination of TDRD3 and PuroR gene were selected for 3 days. Cells were subsequently seeded into 96-well plates at a density of 0.1 cells/well to generate single-cell clones. Only wells harboring a single clone were used. The gene KO was confirmed by Sanger sequencing of the TDRD gene and Western blotting as described below. HuH-7 TOP3B KO cells were generated in the same manner. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.24.005900 doi: bioRxiv preprint Immunoblotting. The HuH-7 wild-type (WT), HuH-7 TDRD3 KO, or HuH-7 TOP3B KO cells were lysed in RIPA buffer (Cell Signaling Technologies) with 1X protease inhibitor cocktail (Cell Signaling Technologies) and centrifuged at 10,000 rpm for 3 min to pellet membranous cell debris. The cleared samples were separated by electrophoresis on a 4 to 12% Bis-Tris protein gels and transferred to nitrocellulose membranes. The membranes were blocked in blocking buffer for The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.24.005900 doi: bioRxiv preprint Cross-liking and Immunoprecipitation (CLIP) of TOP3B. HEK293T-FLP-In Flag-TOP3B was seeded on a 10cm dish at the 2.2 × 10 6 cells/plate and cultured over-night. The culture medium was exchanged to the medium with/without 1 µg/ml DOX to induce exogenous Flag-TOP3B, and at the same time, the cells were infected with DENV-2 at an MOI of 0.1. At the 48 h.p.i., the infected cells were crosslinked with 120 mJ UV light (wavelength 254 nm), or Mocktreated, UV: (-). The cells were lysed with lysis buffer (100mM Tris-HCl pH 7.4, 0.5 % NP-40, 0.5% Triton X-100, 100 mM NaCl 2 , 2.5 mM MgCl 2, and 0.1% sodium dodecyl sulfate (SDS)) and passed through 30G needle. After the centrifuge at 10,000 rpm for 15 min to pellet membranous cell debris, the sample was diluted into 0.25 mg/ml total protein and stored.