Southern blot analysis of recombinant viruses. (A) Schematic genome maps of RVUL4CATgpt, RVwt, RVdlElk-1, RV-137, RV-102, RV-50, and RV-23. The UL4-CAT in its ectopic location is oriented according to the prototype position of the US genes, where transcription is from right to left. The sizes of the DNA fragments resulting from HindIII and SpeI restriction endonuclease digestion are indicated in base pairs. The genes involved in homologous recombination in shuttle vectors are shown in shaded boxes. A, S, and H represent restriction endonuclease sites AvrII, SacI, and HindIII, respectively. (B to E) Autoradiograms of Southern blots to identify the recombinant viruses by using either the ³²P-labeled 110-bp AatII-Avr II DNA (AA probe [B and C]) or HindIII X probe (D and E). Lanes containing viral DNA fragments from different recombinant viruses were spliced together from the same gel. Shuttle vectors pwt-AatII/S, pdlElk-1-AatII/S, and pHBgpt-220CAT AS were used as controls. Panels D and E result from stripping the AA probe from panels B and C, respectively, and then hybridization with the HindIII X probe.

Effect of deletion mutations on CAT expression from the UL4-CAT promoter in infected HFFs. HFFs were infected with recombinant viruses at 5 PFU/cell in quadruplicate as described in the legend to Fig 3. The infection was performed in parallel with that of the RNase protection assay as described in Fig. 3. Equal MOI was confirmed by RNase protection assay of protected IE1 mRNA. The CAT activity per microgram of protein was normalized to IE1 mRNA for each recombinant virus at different time points after infection as described in Materials and Methods. The results of the CAT assays were averaged, and the standard deviations were calculated.

Effect of a MEK inhibitor, UO126, on CAT expression from the UL4-CAT promoter in recombinant virus-infected HFFs. Confluent HFFs were serum starved for 24 h and then treated with UO126 for 1 h before infection with recombinant viruses in the presence of UO126 in quadruplicate as described in Materials and Methods. Cells were harvested at 8 h after infection and subjected to CAT assays. The results of the CAT assays were averaged, and the standard deviations were calculated. The CAT activity per microgram of protein at various concentrations of the drug is relative to that of the nontreated recombinant viruses.

Northern blot analysis of UL4-CAT or UL4 RNA expression in HFFs infected with RVwt in the presence of the MEK inhibitor UO126. Confluent HFFs were serum starved and pretreated with UO126 for 1 h before infection with RVwt as described in the legend to Fig. 6. The drug was present throughout the experiment. Cytoplasmic RNA was harvested at 8 hpi and analyzed by Northern blot hybridization with either the CAT (A), UL4 (B), or actin (C) probe as described in Materials and Methods. The blot was serially stripped and hybridized with different probes. Lanes: 1, untreated; 2, treated with 10 μM UO126; 3, treated with 20 μM UO126.

Schematic representation of the early viral UL4 transcription unit. The UL4 transcription unit in its natural locus drives the synthesis of three unspliced RNAs. Three upstream cellular protein binding sites, 1, 2, and 3, are shown by open, solid, and hatched ovals, respectively. For the E1 (early) viral UL4 promoter, the imperfect dyad NF-Y binding site (CCAAT box) and the TATA box are designated by the solid and open boxes, respectively. The numbers above the symbols are relative to the transcription start site (arrow) of E1.

Steady-state RNA levels transcribed from the UL4-CAT promoter with either the wild type or various mutations at early times after infection. Cytoplasmic RNA was isolated at the indicated time points after HFFs were infected with RVwt, RVdlElk-1, RV-137, RV-102, RV-50, or RV-23 at approximately 5 PFU/cell and then subjected to the RNase protection assay as described in Materials and Methods. (A) Autoradiogram of RNase protection assay. Lanes: 1 to 3, ³²P-labeled CAT, IE1, and actin riboprobes not treated with RNase, respectively; 4, mock infected; 5 to 10, RVwt, RVdlElk-1, RV-137, RV-102, RV-50, and RV-23 at 6 hpi, respectively; 11 to 16, RVwt, RVdlElk-1, RV-137, RV-102, RV-50, and RV-23 at 24 hpi, respectively; 17, ³²P-labeled DNA standard molecular weight markers (Std). For lanes 4 to 10, 150 cpm of both ³²P-labeled IE1 and actin antisense probe was used. For lanes 11 to 16, 150 cpm of both ³²P-labeled actin and CAT antisense probe was used. The sizes of the protected CAT and IE1 RNAs are indicated. nt, nucleotides. (B) Image acquisition analysis of steady-state levels of CAT RNA. The IE1 signal at 6 hpi or the CAT RNA signals at 24 hpi were first normalized to the corresponding actin signals. The resulting numbers were used to determine the final normalized CAT RNA signal. Net cpm, total cpm for an equal area.

Cytotoxicity assays of MAPK inhibitor-treated cells. HFFs in 96-well plates were serum starved for 24 h before treatment with either UO126 for 9 h or FHPI for 15 h in quadruplicate. The cytotoxicity effect of the drug treatment was determined as described in Materials and Methods. (A) Effect of UO126 treatment. (B) Effect of FHPI treatment.

Effect of p38 MAPK inhibitor SB202190 (FHPI) on CAT expression from the UL4-CAT promoter in recombinant virus-infected HFFs. Confluent HFFs were serum starved for 24 h and then treated with FHPI for 1 h before infection with various recombinant viruses in the presence of FHPI in quadruplicate as described in Materials and Methods. Cells were harvested at 14 h after infection and subjected to CAT assays. The results of the CAT assays were averaged, and the standard deviations were calculated. The CAT activity per microgram of protein at various concentrations of the drug is relative to that of the nontreated recombinant viruses.