Figure 1.
pUL11 is a surface expressed glycoprotein.
(A) Predicted structure of the UL11 protein. (B) Flow cytometry with a pUL11-specific rabbit antiserum (black lines) or pre-immune serum (grey lines) of HFF or A549 cells transduced with recombinant adenoviruses expressing UL11 and GFP (rAdV UL11) or GFP alone (rAdV GFP). Gating was on GFP positive cells. (C) Confocal microscopy of HFF and A549 cells transduced as in B) and labeled at the cell surface with the anti-pUL11 serum and an Alexa-568 conjugated anti-rabbit antibody (shown in red). Size bar, 20 µm. (D) Immunoblot with a mouse anti-V5 antibody of lysates of rAdV UL11, rAdV GFP or mock transduced A549 cells. (E) Lysates of rAdV UL11 transduced A549 cells were treated with peptide N-glycosidase F (PNGase F), endo-α-N-acetylgalactosaminidase (O-glycosidase) or untreated as indicated. Immunoblotting was performed as in (D). (D, E) Left margin, molecular mass (in kDa); right margin, the arrowhead and the bracket indicate low and high molecular weight forms of pUL11.
Figure 2.
pUL11 interacts with leukocytes.
(A) Surface staining of the indicated cell lines with purified UL11Fc (black lines), or the Fc control protein (grey lines). (B) Purified UL11Fc or Fc proteins were incubated with primary PBMCs. Surface markers and cell size were used to set gates for different leukocyte subpopulations.
Figure 3.
pUL11 interacts with a T cell surface protein with an approximate mass of 200 kDa.
(A) Lysates of Jurkat or 293T cells, or lysis buffer, were incubated with UL11Fc (UL11) or the Fc control protein (Fc) and protein A sepharose beads. The bound proteins were separated by SDS-PAGE and detected by silver staining. (B) Jurkat or 293T cells were biotinylated prior to lysis. Proteins interacting with UL11Fc, the Fc domain or a CD3ε antibody were precipitated as in (A) and detected after blotting using HRP-streptavidin. A doublet at approx. 200 kDa is indicated.
Figure 4.
(A) Immunoblot with anti-CD45 (upper panel) or anti-CD43 (lower panel) of lysates from 293T and Jurkat (J) cells and proteins precipitated from Jurkat cell lysates with UL11Fc or Fc control protein. Bands corresponding to CD45 and CD43 proteins in Jurkat lysates are indicated. (B) Flow cytometric analysis of the interactions of UL11Fc (black lines, top panel), Fc control protein (grey lines, top panel), CD45 antibody (black lines, middle panel) and UL6Fc (black lines, bottom panel) with Jurkat cells or the Jurkat derived cell lines J-AS-1 and J45.01. Grey lines in middle panels, staining with an isotype-matched control antibody. (C) Binding of UL11Fc (top row, density plots and second row, black lines), Fc control (grey lines) or UL6Fc (third row, black lines) to 293T cells that were mock transfected or transiently transfected with plasmids encoding either human CD45RABC or CD45R0 isoforms, mouse CD45RB or human CD43. Expression of hCD45, mCD45 and hCD43 is shown in density plots in upper panels with rectangles indicating gates for cells analyzed for UL11Fc and UL6Fc binding (rows 2 and 3). 48 h post transfection, cells were incubated with the indicated Fc proteins and co-stained with PE-labeled anti-IgG and FITC-labeled anti-human CD45 or anti-mouse CD45 or anti-human CD43 antibodies. (D) Inhibition of binding of UL11Fc to Jurkat cells by the AICD45.2 CD45 antibody. Cells were incubated with the indicated amounts of AICD45.2 for 30 min, prior to incubation with UL11Fc (black lines) or Fc control protein (grey lines). (E) Interactions of UL11Fc derived from TB40E, Toledo or AD169 strains of CMV (black lines, 3 leftmost columns), or Fc control protein (grey lines, 3 leftmost columns) with Jurkat (top row) or J-AS-1 cells (bottom row). Staining with anti-CD45 (black lines) or an isotype-matched control antibody (grey lines) is shown in the right hand column.
Figure 5.
Surface expressed pUL11 mediates cell adhesion.
(A) PBMCs, Jurkat or J-AS-1 cells were incubated with human fibroblasts that were transduced 3 days earlier with recombinant adenoviruses expressing pUL11 and GFP (rAdV UL11) or GFP alone (rAdV GFP). Unbound cells were removed by washing. White arrows indicate adhering cells, green cells show adenovirus derived GFP. Size bar, 50 µm. (B) Human fibroblasts transduced and depicted as in (A) were left untreated (top row), or incubated for 2 h with anti-UL11 (middle row) or pre-immune rabbit serum (bottom row) prior to incubation with Jurkat cells. Unbound cells were removed by washing. Size bar, 50 µm.
Figure 6.
pUL11 interacts with T cells expressing both long and short isoforms of CD45.
Primary T cells from donors with control or variant CD45 isoform expression were either left untreated (A) or stimulated with phytohaemagglutinin and interleukin-2 (B), and subsequently incubated with UL11Fc (black lines) or the Fc control protein (grey lines) and co-stained with anti-CD45RA or anti-CD45R0 antibodies. Interaction of the Fc proteins is depicted for cells gated as R0 positive, RA positive, or R0 and RA double positive as indicated.
Figure 7.
pUL11 treatment results in reduced T cell signaling and proliferation.
(A) Immunoblot with the 4G10 phosphotyrosine-specific antibody (upper panel) or anti-actin (lower panel) of lysates from Jurkat cells that were either left untreated or were pretreated with UL11Fc or the Fc domain (2.5 µg) for 30 min prior to stimulation with a Jurkat TCR-specific antibody (C305) for the indicated times. (B) Primary T cells were either left untreated (medium) or were incubated with the CD3-specific antibody OKT3 or with phytohaemagglutinin (PHA) together with BSA, UL11Fc or the Fc domain (2 µg) for 3 days. Following incubation for 16 h with 3[H]-thymidine radionucleotide incorporation was measured. All samples were handled in triplicate. Representative data from one of three experiments are shown.