To test if pUL21a was able to bind to the APC in the absence of other HCMV proteins, we performed co-immunoprecipitation assay on lysates from 293T cells transfected with constructs expressing the GFP-amino terminal tagged UL21a (gfpUL21a wt ) or UL21a carrying two stop codons at its amino terminus to abrogate pUL21a expression (gfpUL21a stop ) ( Figure 1D ). Both gfpUL21a wt and gfpUL21a stop were expressed at equal levels but only gfpUL21a wt associated with APC3 or APC8. Additionally, APC3 antibody co-immunoprecipitated gfpUL21a wt but not gfpUL21a stop . We conclude that pUL21a interacts with the APC and does not require other HCMV proteins for this interaction to occur. at an MOI of 5, collected at 48 hpi, and were immunoprecipitated with GFP antibody. Eluted proteins were run on an SDS-containing polyacrylamide gel and silver stained. The bands indicated with an arrow were identified by mass spectrometry as APC3 (100 kDa), APC7, and APC8 (both at 65 kDa). (B) GFP-tagged pUL21a interacts with the APC in HCMV infection. MRC-5 cells were infected as described in panel A, and lysates were subjected to coimmunoprecipitation with GFP or APC3 mouse monoclonal antibodies. Cell lysates and eluted proteins were analyzed by immunoblotting with indicated antibodies. GFP blots were cropped to save space but were from the same lane and exposed film. Non-specific cross-reacting bands are indicated by asterisk (see text). Partial proteolysis was often seen with the GFP-tagged UL21a protein, particularly in cell lysate samples. (C) Native pUL21a interacts with the APC in HCMV infection. MRC-5 cells were infected with ADgfp or ADsubUL21a (as described in Materials and Methods) in the presence (+) or absence (2) of 10 mM MG132. Cell lysates were prepared at 24 hpi and immunoprecipitated with APC3 antibody. Cell lysates and eluted proteins were analyzed by immunoblotting. (D) Interaction of pUL21a with the APC does not require other viral proteins. 293T cells were transfected with plasmids expressing gfpUL21a wt or gfpUL21a stop . Cells were collected 72 hours later and cell lysates were immunoprecipitated as in panel B. Cell lysates and eluted proteins were analyzed by immunoblotting. PCNA and pUL44 were used as cellular and viral negative controls, respectively. doi:10.1371/journal.ppat.1002789.g001