PMC

Localization of the UL11 proteins within CMV-infected cells. (A) RPE-1 cells either mock infected or infected with the indicated viruses were labeled with a UL11-specific MAb (black line) or the isotype control Ab (gray shading) and analyzed by flow cytometry. max, maximum. (B) Human fibroblasts infected with the HA11GM mutant were stained at 72 h p.i. with DAPI, and Abs specific for calnexin, Golgi compartment-resident protein p230, and EEA1 and fluorescence were visualized by confocal microscopy. Bars, 25 μm.

Posttranslational modification and surface expression of UL11 proteins. (A) Lysates of HM11DL-infected (lane 1) and HM11V5-infected (lanes 2 to 11) RPE-1 cells were subjected to immunoprecipitation with a V5-specific mouse Ab, and the eluted fractions were treated with endo H, PNGase F, O-glycosidase, neuraminidase, or a mix of deglycosylating enzymes or left untreated, as indicated, followed by immunoblotting with a V5-specific rabbit Ab. (B to E) Surface proteins of cells infected with the HM11V5, HM11DL, HA11G, or HA11D mutant were labeled with biotin and subjected to precipitation with streptavidin-coated beads, followed by immunoblotting with Abs specific for the V5 tag, GFP, p230, or EGFR. Input lanes show proteins of the whole-cell lysate before precipitation. The highly glycosylated UL11 form is marked with an asterisk, and the band resulting from the unspecific reactivity of the GFP Ab is indicated with a circle. The sizes of the marker bands (indicated to the left or right of each panel) are in kDa. PD, pulldown.

Expression of ORF UL11-encoded proteins in CMV-infected cells. (A) Lysates of HM11SF- or HMpar-infected cells were subjected to precipitation with streptactin-coated agarose beads, followed by immunoblotting with a FLAG tag-specific antibody. (B, C) Lysates of HM11V5-, HM11V5S-, or HM11DL-infected RPE-1 cells, which were kept with (+) or without (−) the proteasomal inhibitor MG-132, were subjected to immunoprecipitation and immunoblotting with a V5-specific antibody. High- and low-molecular-mass UL11 bands are indicated with asterisks and closed circles, respectively. Prior to immunoprecipitation, lysates were probed with an antibody specific for the CMV UL44 protein, serving as an infection and loading control. The reactivities of the heavy chain (HC) and light chain (LC) of the V5 Ab are indicated. (D) Human fibroblasts infected with the HA11G or HA11D mutant were harvested on the indicated day postinfection (dpi) and subjected to immunoblotting with a GFP-specific (top), an IE1-specific (middle), or a UL44-specific (bottom) Ab. UL11-specific bands are indicated as described in the legend to panel A, and the nonspecific reactivity of the GFP Ab is marked with open circles. The asterisks and closed circle are as described in the legend to panels B and C. The sizes of the marker bands (indicated to the left of each panel) are in kDa.

CMV mutants and lentiviral constructs used in this study. The scheme depicts the structure of the CMV genome (top) with the terminal repeat (TR), internal repeat (IR), unique long (UL), and unique short (US) sequences. The genomic region with the UL11 ORF and the genetic elements added, deleted (Δ), or replaced in the mutants with the indicated names are shown enlarged below. mGFP, monomeric green fluorescent protein; mMIEP, mouse CMV major immediate early promoter; SF, Strep-FLAG tag; V5, V5 epitope tag; TGA, replacement of the UL11 start codon with the stop codon TGA; Gluc, Gaussia luciferase. The mutants are based on the AD169 or the Merlin strain. The lentiviral vector Sm UL11 contains the long terminal repeats (LTRs), the human CMV MIEP (hMIEP), and the ORFs for the V5-tagged UL11 and for GFP separated by an internal ribosome entry site (IRES) element. The Sm CTRL vector encoding only GFP was used as a control.

UL11 expressed in HEK293T cells does not affect activation of CMV-specific CD8 T cells. (A) Lysates of untreated HEK293T cells (lane 1) or cells transduced with lentiviral vectors Sm CTRL (lane 2) or Sm UL11 (lane 3) were subjected to immunoblotting with the UL11-specific monoclonal antibody (mAb). The highly and less glycosylated forms of UL11 are indicated with asterisks. The GAPDH signal served as a loading control. The sizes of the marker bands (indicated to the left) are in kDa. (B) Flow cytometric analysis of transduced HEK293T cells stained with the isotype control (gray shading) or the UL11-specific Abs (black line). (C) Untreated HEK293T cells or cells transduced with the indicated lentiviral vectors were preincubated with the IE1 protein-derived peptide CRVLCCVML (CRV) or VLEETSVML (VLE) and cocultivated with the indicated specific CD8 T cell clones. T cell reactivity was analyzed by measuring the overnight IFN-γ secretion by ELISA. Bars show the means ± SDs for triplicate samples. For clones F46M#160 CRV and F61M#35 VLE, one result of three independent experiments is shown, and for clones LTM#131 CRV and LTM#17 VLE, one result of two independent experiments is shown. Statistical analysis (two-tailed Mann-Whitney test) did not indicate significant differences. n.s., not significant.

Analysis of UL11 expressed in CMV-infected fibroblasts on activation of CMV-specific T cell clones. (A) CD45 expression (left) and binding of the soluble UL11-Fc protein to the indicated CD8 T cell clones (right) were determined by flow cytometry upon labeling with a CD45 Ab (black line) or an isotype control Ab (gray shading) and upon incubation with the UL11-Fc protein (black line) or the Fc control protein (gray shading). (B) Immunoblot analysis for IE1 and pp65 proteins of MRC-5 cells infected with the indicated viruses at an MOI of 2 for 78 h. GAPDH served as a loading control. (C) MRC-5 cells either mock infected or infected with the indicated viruses, as described in the legend to panel B, were examined for surface expression of HLA class I molecules by flow cytometry, and mean fluorescence intensity (MFI) values are depicted. (D) MRC-5 cells infected as described in the legend to panel B were labeled with an antibody against the major capsid protein (MCP), and nuclei were stained with DAPI. Five random images were taken per setting, and the percentages of MCP-positive cells were determined. (E) MRC-5 cells infected with the HA11G or HA11D mutant were cocultivated with CD8 T cell clones specific for the CRVLCCVML (IE1-derived) or NLVPMVATV (pp65-derived) peptide. T cell reactivity was analyzed by measuring overnight IFN-γ secretion by ELISA. Mock-infected cells and cells loaded with peptides (pep) were used as negative and positive controls, respectively. Data show the means ± SDs for triplicate samples.