[ 3 H]uridine labeling of MHV RNA Cells plated in 6-well plates were infected with MHV-A59 at an m.o.i. of 2. At 1 h p.i., 5 mg/ml actinomycin D was added to the virus growth medium to inhibit cellular RNA synthesis. To label newly synthesized MHV RNA, 100 mCi/ml of [ 3 H]uridine (NEN, Boston, MA) were added to the medium at hourly intervals. After 1 h of labeling, the cells were washed twice in ice-cold PBS and scraped off the plates in 1 ml of PBS. The cells were then collected by centrifugation and incubated in 200 ml of NTE buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA) containing 0.5% NP-40, 0.5 mM dithiothreitol (DTT) and 400 U/ml of RNasin on ice for 15 min. After centrifugation, 5 ml of the cytoplasmic extract were spotted on a piece of 3 mm paper and incubated with 5% trichloroacetic acid (TCA). The radioactivity remaining on the 3 mm paper was measured in a scintillation counter.