MCMV gp48-mediated trafficking of class I MHC molecules is affected by depletion of AP-1μ or AP-3μ subunits.

<p>Immunofluorescence analysis of class I MHC molecules in cells expressing gp48HA, − (a) and + (b) AP-1μ siRNA, and - (d) and + (e) AP-3μ siRNA, as indicated. Arrows in panels b and e point to the plasma membrane. Scale bar = 10 µm. c and f) Immunoblot analysis of AP-1μ or AP-3μ from lysates of U373-gp48HA cells −/+ treatment with AP-1μ (c) or AP-3μ (f) siRNA, as indicated. The Ponceau S stained nitrocellulose is shown beneath the immunoblots as a loading control.</p

U21 can reroute class I MHC molecules in the absence of AP2µ.

<p>a) Schematic representation of the AP-2 complex (redrawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099139#pone.0099139-Bonifacino1" target="_blank">[15]</a>). b) AP2µ immunoblot of lysates from U373-U21 cells before and 5 days after introduction of AP-2µ shRNA. The Ponceau S stained nitrocellulose is shown beneath the immunoblot as a loading control. c) Flow cytometric analysis of class I MHC molecules on the cell surface of U373 or U373-U21 cells, 5 days after introduction of AP-2µ shRNA. Cell lines − (red) and + (blue) AP-2µ shRNA are indicated. d,e) U373 and U373-U21 cells or AP2µ shRNA-expressing U373 and U373-U21 cells were labeled with W6/32, directed against properly-folded class I MHC molecules, as indicated. Arrows in panel d point to the plasma membrane. f,g) U373-U21 cells were double-labeled with W6/32 and anti-lamp2 Arrows point to specific puncta that overlap. h). The images are shown overlayed in (h), with class I molecules in red, and lamp2 in green, as indicated. Cells are shown at the same magnification. Scale bar = 10 µm.</p

Rescue of AP-3 depletion.

<p>a) anti-AP-3μ immunoblot analysis of lysates from U373-U21 cells (lanes 1,2) or U373-U21 cells expressing FLAG-AP-3μ^rescue (lanes 3,4), −/+ AP-3μ shRNA, as indicated. The Ponceau S stained nitrocellulose is shown beneath the immunoblots as a loading control. b) Flow cytometric analysis of surface class I MHC molecules on U373-U21 or U373-U21 cells expressing FLAG-AP-3 µ^rescue, −/+ AP-3μ shRNA depletion, as indicated.</p

Tailless U21 is affected by depletion of adaptor complexes.

<p>Immunofluorescence analysis of class I MHC molecules in tailless-U21-expressing cells (U21NT), − (a) and + (b) AP-1μ shRNA expression, as indicated. Arrows in panel b point to the plasma membrane. Scale bar = 10 µm.</p

siRNA-mediated depletion of AP-1 and AP-3.

<p>a) anti-AP-1μ or anti-AP-3μ immunoblot analysis of lysates from U373-U21 cells −/+ siRNA mediated depletion, as indicated. The Ponceau S stained nitrocellulose is shown beneath the immunoblots as a loading control. b,c) Flow cytometric analysis of class I MHC molecules on the cell surface of U373-U21 cells, −/+ AP-1μ or AP-3µ siRNA. Cell lines − (red traces) and + (blue traces) AP-1μ (b) or AP-3µ (c) siRNA are indicated. d) Cell surface levels of the transferrin receptor (TfR) in U373-U21 cells −/+ siRNA-mediated depletion of AP1μ, as indicated.</p

U21 does not reroute class I MHC molecules in the absence of AP3µ.

<p>a) Schematic representation of the AP-3 complex (redrawn from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099139#pone.0099139-Bonifacino1" target="_blank">[15]</a>). b) AP-3µ immunoblot of lysates from U373-U21 cells before and 5 days after introduction of AP-3µ shRNA. The Ponceau S stained nitrocellulose is shown beneath the immunoblot as a loading control c) Flow cytometric analysis of class I MHC molecules on the cell surface of U373 or U373-U21 cells, 5 days after introduction of AP-3µ shRNA. Cell lines − (red) and + (blue) AP-3µ shRNA are indicated. d,e) U373 and U373-U21 cells or AP-3µ shRNA-expressing U373 and U373-U21 cells were labeled with W6/32, as indicated, 5 days after introduction of AP-3µ shRNA. Arrows in panel e point to the plasma membrane. Scale bar = 10 µm.</p

Natural Killer Cell Evasion Is Essential for Infection by Rhesus Cytomegalovirus

<div><p>The natural killer cell receptor NKG2D activates NK cells by engaging one of several ligands (NKG2DLs) belonging to either the MIC or ULBP families. Human cytomegalovirus (HCMV) UL16 and UL142 counteract this activation by retaining NKG2DLs and US18 and US20 act via lysomal degradation but the importance of NK cell evasion for infection is unknown. Since NKG2DLs are highly conserved in rhesus macaques, we characterized how NKG2DL interception by rhesus cytomegalovirus (RhCMV) impacts infection <i>in vivo</i>. Interestingly, RhCMV lacks homologs of UL16 and UL142 but instead employs Rh159, the homolog of UL148, to prevent NKG2DL surface expression. Rh159 resides in the endoplasmic reticulum and retains several NKG2DLs whereas UL148 does not interfere with NKG2DL expression. Deletion of Rh159 releases human and rhesus MIC proteins, but not ULBPs, from retention while increasing NK cell stimulation by infected cells. Importantly, RhCMV lacking Rh159 cannot infect CMV-naïve animals unless CD8+ cells, including NK cells, are depleted. However, infection can be rescued by replacing Rh159 with HCMV UL16 suggesting that Rh159 and UL16 perform similar functions <i>in vivo</i>. We therefore conclude that cytomegaloviral interference with NK cell activation is essential to establish but not to maintain chronic infection.</p></div

Deletion of Rh159 rescues intracellular transport and surface expression of MICA and MICB upon RhCMV infection.

<p><b>A)</b> Comparison of NKG2DL surface expression upon infection with RhCMV or ΔRh159. U373-NKG2DL cells were infected with RhCMV (blue) or ΔRh159 (red) (MOI = 3) for 48 h. Cell surface levels of NKG2DL or TfR were determined by flow cytometry, using specific antibodies and compared to isotype control (dotted). Depicted is NKG2DL or TfR surface expression on infected cells gated for RhCMV IE2⁺ expression. The results shown are representative of three or more independent experiments. <b>B)</b> Biosynthesis and maturation of NKG2DL in uninfected U373-NKG2DL cells or upon infection with RhCMV or ΔRh159. U373-NKG2DL cells were uninfected (NI), infected with RhCMV (WT) or ΔRh159 (MOI = 3) for 24 h, verified by light microscopy as having 100% CPE, then metabolically labeled with [35S]cysteine and [35S]methionine for 30 min prior to chasing the label for the indicated times. The indicated NKG2DLs were immunoprecipitated from cell lysates with specific mAbs. Immunoprecipitates were split and digested with EndoH (+) or mock treated (-) then analyzed by SDS-PAGE and autoradiography.</p

Rh159 interferes with intracellular transport of NKG2DL.

<p><b>A)</b> Association with Rh159 prevents intracellular transport of MICB. U373-MICB cells were transduced with adenovectors (MOI = 80) expressing either GFP (AdGFP) or FLAG-tagged Rh159 (AdRh159FL) under control of tetracycline-dependent transactivator provided by co-transduced AdtTA (MOI = 20). At 24 hpi cells were metabolically labeled for 30 min with [35S]cysteine + [35S]methionine. Upon chasing the label for the indicated times (h), cells were lysed and MICB was immunoprecipitated with anti–MICB mAb. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. (S) indicates EndoH-deglycosylated proteins. <b>B)</b> Rh159 co-immunoprecipitates with MICB. U373-ULBP3 (ULBP3, left panel) or U373-MICB (MICB, right panel) cells were lysed at 48 h post-transduction with AdRh159FL (Rh159) or an adenovector expressing FLAG-tagged SVV ORF 61 (SVV61) used as a negative control. MICB and ULBP3 were immunoprecipitated with anti–MICB and anti-ULBP3 mouse and goat mAbs, respectively, then immunoblotted with mouse anti-FLAG mAb. The mouse IgG heavy chain (55kDa) is indicated (HC). Input lanes were loaded with 10% total lysate used in immunoprecipitation and immunoblotted with mAbs for FLAG and GAPDH. The results shown are representative of two independent experiments. <b>C)</b> Rh159 reduces steady state levels of MICB. U373-MICB cells were lysed at 48 h post-transduction with the indicated Ad-vectors. Lysates were digested with EndoH (+) or mock treated (-) then immunoblotted with mAbs for MICB, FLAG or GAPDH. Note that both MICB and Rh159 are EndoH sensitive consistent with ER localization. The results shown are representative of two independent experiments. <b>D-E)</b> Rh159 reduces surface expression of MICA, MICB, ULBP1 and ULBP2 but not ULBP3. U373-NKG2DL cells were transduced with AdRh159FL or AdGFP as in A) but for 48 h. Cells were then lysed and immunoblotted with mAbs for FLAG and GAPDH (<b>D</b>), or stained with antibodies specific for the indicated proteins, or isotype control (dotted) and analyzed by flow cytometry. The results shown are representative of three or more independent experiments.</p

Primary infection of rhesus macaques requires evasion of NK cells by Rh159.

<p><b>A)</b> Rh159 is required for superinfection. At day 0, a RhCMV+ RM was infected subcutaneously (s.c.) with 5x10⁶ PFU of ΔRh159. The SIVgag-specific T cell response in PBMC was monitored by ICCS for CD69, TNFα and IFNγ using overlapping (by 4AA) 15mer peptide mixes. Results are shown as a percentage of total memory CD4⁺ or CD8⁺ T cells. No responses above background were measured. <b>B)</b> Rh159 is required for primary infection. Two RhCMV-naïve animals were inoculated s.c. with 5x10⁶ PFU ΔRh159 and SIVgag- and RhCMV IE-specific T cells were monitored as in A). In addition, SIVgag-specific CD8+ T cell responses to 2 MHC-E restricted (Gag69 and Gag120) and 2 MHC-II-restricted (Gag53 and Gag73) supertope peptides were quantified by flow cytometric ICS. Starting on day 63, RM were treated with anti-CD8 antibody CM-T807 to deplete CD8⁺ cells and RM were re-inoculated with 5x10⁶ PFU of ΔRh159 on day 64. <b>C)</b> The relative frequencies of CD8⁺ small CD3- lymphocytes in whole blood (WB) of each animal were monitored during CM-T807 treatment.</p