FIG. 4.

Steady-state RNA levels transcribed from the UL4-CAT promoter with either a wild-type or mutant NF-Y binding site at early times after infection. Cytoplasmic RNA was isolated at the indicated time points after infection with RVwt-xs or RVdlNF-Y-xs and then subjected to RNase protection assays as described in Materials and Methods. (A) Autoradiogram of RNase protection assay. Lanes: 1, ³²P-labeled DNA standard molecular weight markers; 2 to 4, ³²P-labeled CAT, actin, and IE1 riboprobes not treated with RNase, respectively; 5, mock infected; 6, RVwt-xs at 6 hpi; 7, RVdlNF-Y-xs at 6 hpi; 8, RVwt-xs at 24 hpi; 9, RVdlNF-Y-xs at 24 hpi; 10, RVwt-xs at 48 hpi with PAA treatment; 11, RVdlNF-Y-xs at 48 hpi with phosphonoacetic acid (PAA) treatment. The sizes of the protected RNAs are indicated (in nucleotides [nt]). Lanes 6 and 7, both the CAT and IE1 probes were added to the reaction mixture. Lanes 5 and 8 to 11, both the CAT and actin probes were added to the reaction mixture. (B) Image acquisition analysis. The CAT RNA signals from RVwt-xs and RVdlNF-Y-xs was normalized to protected IE1 (6 hpi) or actin (24 hpi, 48 hpi + PAA) RNA.