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and U373-MG astrocytoma (U373) control cells were as described (20). Both the US2+ and US3+ cell line used in this study, originally designated 55–310 cells and 5–214 cells (respectively) in laboratory records, were derived from U373 cells. To obtain these cell lines, HCMV US2 and US3 genes were cloned into plasmids such that their expression was under the control of the HCMV major immediate-early promoter. The US2 open reading frame was cloned as a 0.776-kb BanII-XhoI DNA fragment (bases 193,779–193,003) in vector pIEsp-puro (20), to yield pIEspUS2-puro. The US3 open reading frame was cloned as a 0.638-kb AvaII-SacII DNA fragment (bases 194,700–194,062) in vector pIE-puro (20), to yield pIEpuro-US3(AS). Stably transfected US2+ and US3+ cell lines were obtained via transfection of these plasmids and selection in puromycin-containing medium as described (20).

Glutathione S-Transferase (GST)-US3 Fusion Protein. A GST-US3 fusion protein was made using a modified vector, pGST-Nco (provided by I.Mohr, Wyeth-Ayerst Research, Pearl River, NY). Using the polymerase chain reaction, the region of the US3 gene representing the region encoding amino acids 33–112 (22) was amplified in a manner that resulted in the insertion of a 5′ NcoI and a 3′ EcoRI sites to facilitate directional cloning between those same sites in pGST-Nco vector. After transfection into Escherichia coli DH5, expression of the GST-US3 fusion protein was induced with isopropyl β-D-thiogalactoside. After sonication of the induced bacteria, the fusion protein was located in the pellet fraction, then solubilized, and electroeluted from SDS/ polyacrylamide gels.

Antibodies. Rabbit polyclonal antisera reactive with HCMV US2 and US11 proteins have been described (ref. 20; T.R.J. and L.S., unpublished results). Polyclonal antisera reactive with HCMV US3 protein was derived from New Zealand White rabbits immunized with the GST-US3 fusion protein (Cocalico Biologicals, Reamstown, PA). Rabbit polyclonal antisera designated anti-HC, which reacts free MHC class I heavy chains, has been described (17). Murine monoclonal antibodies W6/32 (28), specific for a conformation-dependent epitope on the heavy chain of human MHC class I proteins, and Ber-T9, specific for the human transferrin receptor, were purchased from Dako. Murine monoclonal antibody TP25.99 (29), specific for a conformation-independent epitope on MHC class I heavy chains, was obtained from S.Ferrone (New York Medical College, Valhalla, NY). BBM.1 monoclonal antibody recognizes both free and heavy chain-associated human β2-microglobulin (30). Anti-vertebrate actin monoclonal antibody clone C4 was purchased from Boehringer Mannheim.

Metabolic Labeling, Immunoprecipitation, and Immunoblot Analysis. Metabolic labeling, immunoprecipitation, and immunoblot techniques were done as described (20). Where indicated, the mild detergent digitonin (Boehringer Mannheim) was used for cell lysis (1% final concentration) and in all immunoprecipitation steps (0.2% final concentration). Digitonin was dissolved in buffer containing 25 mM Hepes (pH7.2)/10 mM CaCl2. For pulse-chase experiments, 1 ml of complete medium containing 2× unlabeled methionine/ cysteine was added directly to the radioactive pulse medium and incubation was continued until the proper harvest time. Radioiodination of cell surface proteins was done with sodium 125I (New England Nuclear) according to standard protocols (31). Endoglycosidase H and N-glycosidase F digestions were done for 18 hr at 37°C after immunoprecipitation using the recommended conditions (Boehringer Mannheim).

Immunofluorescence Microscopy. In some experiments, expression of MHC class I molecules was detected by immunofluorescence microscopy as described (20), except that the blocking step with human serum was omitted. Fixed cells were treated sequentially with TP25.99 primary antibody and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Pierce) secondary antibody. Confocal immunofluorescence microscopy was performed as described (32), except that TP25.99 monoclonal antibody was used to detect MHC class I heavy chains. The dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6[3]) (Molecular Probes) was used to stain the ER as described (33). These samples were visualized using a Leica confocal laser scanning microscope. Class I heavy chain fluorescence was quantified using the QUANTIMET 500 fluorescence analysis program (Leica).

RESULTS

Expression and Characterization of US3 Gene Products. The open reading frames of the HCMV US2 and US3 genes

FIG. 1. Genome location and expression from HCMV US3. (A) Relative location of US2, US3, and US11 in the HCMV genome. (B) Uninfected US3+ cells or U373 control cells were metabolically radiolabeled for 2 hr (lanes P) then chased in medium containing excess unlabeled amino acids for 3 hr (lanes C). Human foreskin fibroblasts were infected with either HCMV wild-type AD169 or mutant RV47 at a multiplicity of infection of 3 and radiolabeled from 3 to 7 hr after infection. Immunoprecipitation was done with US3 antibody. (C) US3+ cells were metabolically radiolabeled in pulse-chase fashion as indicated prior to immunoprecipitation, using US3 antibody, and glycosidase treatment. For control purposes, an immunoprecipitation from pulse-labeled U373 control cells is shown. gpUS3, 22-kDa US3 glycoprotein; *, 18-kDa US3 product; **, deglycosylated US3 protein; EH, endoglycosidase H digestion; NG, N-glycosidase F digestion.



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