Fig. 3.

Identification of pUL138 as an enhancer of TNFR1 surface density. (A) Scheme of AD169-ULb′ ORFs. Candidate ORFs for TNFR1 upregulation (present in AD169varL and absent from Towne) are depicted in gray. (B) Subcellular localization of ectopically expressed HA-tagged ULb′ candidate proteins. HeLa cells were transfected with the respective expression plasmid. Twenty-four h posttransfection, cells were analyzed by immunofluorescence staining (anti-HA). (C) pUL138 mediates the upregulation of TNFR1 surface expression. HeLa cells were transfected with pIRES-UL138HA-EGFP. Twenty-four h posttransfection, cells were harvested for analysis by flow cytometry. DAPI-negative cells of GFP-positive and GFP-negative gates were analyzed for TNFR1 surface density. TNF-α treatment (20 ng/ml for 30 min) before staining served as a specificity control of TNFR1 detection, since ligand binding competes with MAB225 binding. (D to G) MRC-5 fibroblasts were mock treated or infected with AD169varL (MOI, 5). (D) Cells were harvested for RNA preparation at the indicated times. Equal amounts of total RNA were separated on a MOPS gel, and UL138 transcripts were detected by Northern blotting. (E) Selective transcription of HCMV immediate-early genes was achieved by infection in the presence of cycloheximide (CHX; 50 μg/ml). (F) HCMV DNA replication was prevented by the use of ganciclovir (GCV; 50 μM) to determine GCV-sensitive transcripts. Infected cells were harvested for RNA preparation at the indicated times. Equal amounts of total RNA were separated on a MOPS gel, and UL138-spanning transcripts were detected by Northern blotting. (G) Cells were harvested at the indicated times for the analysis of TNFR1 surface expression by flow cytometry. Histograms of DAPI-negative cells are shown.