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

We thank Robert Silverman, Gokhan Hotamisligil, and Anthony Sadler for PKR mutant 610 and wild type cells. We thank Katelyn Green, Becky Billmire, and Peter Todd for the use of and 611 assistance with the Brandel BR-188 Density Gradient Fractionation System and for providing the 612 nanoluciferase and firefly luciferase plasmids. We thank Zachary Mendel and Joel Swanson for 613 assistance with imaging the DQ BSA assay. We thank Jason Weinberg, Michael Imperiale, 614 The cell pellets were collected and RNA was isolated. cDNA was generated from the RNA, and 904 qPCR was used to quantitate PKR mRNA levels. Each graph contains 5-7 replicates for each with MAV-1 (MAV) at an MOI of 5 or mock infected (mock) and collected at 48 hpi. Cells were 909 lysed, and cleared lysates from three 10 cm plates were layered onto 25% sucrose and 910 centrifuged to pellet ribosomes. RNA was purified from the pellets, cDNA was generated from 911 the RNA, and qPCR was used to quantitate the PKR mRNA levels. The graph contains 9 912 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 https://doi.org/10.1101/584680 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 https://doi.org/10.1101/584680 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 MAV-1 (MAV) at an MOI of 5 or mock infected (mock) and collected at 48 hpi. Cells were lysed, and cleared lysates from three 10 cm plates were layered onto 25% sucrose and centrifuged to pellet ribosomes. RNA was purified from the pellets, cDNA was generated from the RNA, and qPCR was used to quantitate the PKR mRNA levels. The graph contains 9 replicates for each time point, The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 https://doi.org/10.1101/584680 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. Figure 5 . PKR is not detectably ubiquitinated during MAV-1 infection. CMT93 cells were transfected with a GFP-ubiquitin plasmid using standard Polyplus transfection protocols. At 24 hpt, the cells were infected with MAV-1 (MAV) at an MOI of 5 or mock infected (mock) and treated with 10 µM MG132 at 6 hpi. Cell lysates were collected at 12 hpi and immunoprecipitated with (A) PKR (D-20) or an isotype control antibody or (B) p53 (DO-1) or an isotype control antibody. Immunoprecipitated samples were analyzed by immunoblot with GFP, ubiquitin, isotype, PKR (B-10), or p53 (1801) 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 Supplemental Figure 6 . Viral DNA replication can be detected at 24 hpi by qPCR. CMT93 cells were infected with MAV-1 (MAV) at an MOI of 10 and collected every 6 hours for 24 hours. DNA was purified from cell pellets and analyzed for MAV-1 genome copies by qPCR. Graph is representative of four to five biological replicates per treatment group. Error bars are standard error of the mean (SEM). 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