Reovirus Activates a Caspase-Independent Cell Death Pathway

Berger, Angela K.; Danthi, Pranav

Murine L929 cells (ATCC CCL-1) were maintained in Eagle's minimal essential medium (MEM) (Lonza) supplemented with 5% fetal bovine serum (FBS) and 2 mM L-glutamine. Spinner-adapted L929 cells (obtained from T. Dermody's laboratory) were maintained in Joklik's MEM (Lonza) supplemented to contain 5% FBS, 2 mM L-glutamine, 100 U/ml of penicillin, 100 g/ml of streptomycin, and 25 ng/ml of amphotericin B. Spinner-adapted L929 cells were used for cultivating and purifying viruses and for plaque assays. ATCC L929 cells were used for all experiments to assess cell death and cell signaling. No differences were observed in permissivity between ATCC L929 cells and spinner-adapted L929 cells. Prototype reovirus strains T1L and T3D were regenerated by plasmid-based reverse genetics (77, 78) . No reverse genetic system is available for T3A, and thus, a laboratory stock was used. Infectious and genome-deficient viral particles were purified by Vertrel XF extraction and CsCl gradient centrifugation (79) . Viral titer was determined by a plaque assay using spinner-adapted L929 cells (80) . UVinactivated virus was generated using a UV cross-linker (CL-1000 UV Crosslinker; UVP). T3D virus (1 Ï« 10 9 PFU/ml) was irradiated with short-wave (254-nm) UV on ice at a distance of 10 cm for 1 min at 120,000 J/cm 2 in a 60-mm tissue culture dish. Particle numbers for top-component and infectious virus were determined by estimating the protein concentration in each sample. One hundred eighty-five micrograms of protein was considered equal to 2.1 Ï« 10 12 virions (81). Relative per-particle infectivity of intact, genome-deficient virions and UVinactivated virions was determined using indirect immunofluorescence.