Enhanced replication of mouse adenovirus type 1 following virus-induced degradation of protein kinase R (PKR)

Danielle E. Goodman; Carla D. Pretto; Tomas A. Krepostman; Kelly E. Carnahan; Katherine R. Spindler

MAV-1 at an MOI of 5. C57BL/6 MEF lysates were collected at 48 hpi by scraping the cells in 571 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/584680 doi: bioRxiv preprint ice-cold PBS containing 100 µg/mL cycloheximide (Sigma C7698), pelleting, and resuspending 572 in lysis buffer, which was 10 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 4 mM DTT, 573 0.5% NP-40, 100 µg/mL cycloheximide, 20 U/mL RNasin (Promega # N2511), 10% sucrose, 574 and 1x protease inhibitors (Protease Inhibitor Cocktail Kit, Thermo Scientific #78410). Cells 575 were lysed by passage through a chilled 26G needle five times and cleared by centrifugation for 576 10 min at 21,000 x g at 4˚C. 400 µL of cleared lysate (10 OD 260nm units) was layered onto 25% 577 sucrose and centrifuged 29,500 rpm in an SW41 rotor (107,458 rcf average) for 4 hours at 4˚C. (ThermoFisher Scientific Cat# AM2238), and 1% Triton X-100 (Sigma T9284). Cells were lysed 594 by passaging through a chilled 26G needle ten times, vortexing for 30 seconds, and then 595 incubating on ice for 5 min. Lysates were cleared by centrifugation for 5 min at 14,000 x g at 596 4˚C. 500 µL of cleared lysate (10 OD 260nm ) was layered onto a 10-50% sucrose gradient and 597 centrifuged at 35,000 rpm in an SW41 rotor (151,000 rcf) for 3 hours at 4˚C. After 598 centrifugation, gradients were pumped out of the top with a Brandel BR-188 Density Gradient 599