Comparison of gene expression profiles between GagCM9⁺cells_SIV+RMs and in CD8⁺cells_SIV+RMs vs. CD8⁺cells_SIV-RMs.

<p><b>A)</b> The graph shows the distribution and median of the averaged transformed Log₂ gene expression showing that the overall gene expression intensity by animal and sample type is similar in all groups. <b>B)</b> The graph shows a positive correlation (r² = 0.70) of gene expression differences between GagCM9⁺cells_SIV+RMs (Y-axis) and CD8⁺cells_SIV+RMs (X-axis) relative to CD8⁺cells_SIV-RMs. Genes were filtered by those differentially expressed genes found when comparing GagCM9⁺cells_SIV+RMs with CD8⁺cells_SIV-RMs or CD8⁺cells_SIV+RMs with CD8⁺cells_SIV-RMs followed by FC calculations. The blue filled circles symbolize the differently expressed genes in at least one comparison between GagCM9⁺cells_SIV+RMs and CD8⁺cells_SIV+RMs to that of CD8⁺cells_SIV-RMs control (p<0.05). <b>C)</b> The Venn diagram illustrates how the 2346 genes, which were significantly altered by SIV infection (based on a significance cutoff threshold of p<0.05) overlap between the three comparisons; GagCM9⁺cells_SIV+RMs vs. CD8⁺cells_SIV+RMs, (blue circle); GagCM9⁺cells_SIV+RMs vs. CD8⁺cells_SIV-RMs (red circle); CD8⁺ cells_SIV+RMs vs. CD8⁺cells_SIV-RMs (green circle).</p

Laser capture microdissection of GagCM9⁺cells_SIV+RMs, CD8⁺cells_SIV+RMs and CD8⁺cells_SIV-RMs.

<p><b>A)</b> Fluorescence images of lymph node tissue sections from an SIV infected RM stained with GagCM9 Qdot 655 multimer (red), CD8 (green) and dapi (blue) showing the abundance of GagCM9⁺ cells. <b>B)</b> A magnified view of the region indicated in panel A, demonstrating that the GagCM9⁺cells_SIV+RMs cells also express CD8⁺. <b>C)</b> Images of lymph node tissue sections from an SIV infected RM stained with CM9 Qdot 655 multimer to detect GagCM9⁺cells_SIV+RMs. The left images show the tissue section with the selected GagCM9⁺cells_SIV+RMs prior to LCM and the images to the right show the same tissue section after the selected cells have been microdissected and collected.</p

The top 10 differentially over/under-expressed genes between GagCM9⁺cells_SIV+RMs vs. CD8⁺cells_SIV+RMs.

<p>The top 10 differentially over/under-expressed genes between GagCM9⁺cells_SIV+RMs vs. CD8⁺cells_SIV+RMs.</p

Heat map of top five up and downregulated differentially expressed genes.

<p>The heat map shows discrimination of the gene profiles of top five up and downregulated differentially expressed genes in the comparison between <b>A)</b> GagCM9⁺cells_SIV+RMs vs. CD8⁺cells_SIV+RMs and <b>B)</b> GagCM9⁺cells_SIV+RMs vs. CD8⁺cells_SIV-RMs. Gene expression levels are shown in color, with red indicating over-abundant expression and blue indicating under-abundant expression.</p

The top 10 differentially expressed genes between CD8⁺cells_SIV+RMs vs. CD8⁺cells_SIV-RMs.

<p>The top 10 differentially expressed genes between CD8⁺cells_SIV+RMs vs. CD8⁺cells_SIV-RMs.</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

Cell surface expression of NKG2DLs is not affected by HCMV UL148.

<p><b>A)</b> Alignment of Rh159 and UL148. Intensity of purple shading indicates level of sequence conservation (Jalview 2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005868#ppat.1005868.ref055" target="_blank">55</a>]). <b>B</b>) MICB maturation is not affected by UL148. U373-MICB cells were transduced with AdCtrl (empty vector) (Ctrl), or AdUL148 containing a C-terminal V5 tag (UL148) at an MOI of 10 or 80, or AdRh159FL (Rh159) (MOI = 10) together with AdtTA (MOI = 2.5) for 48 h. UL148 and Rh159 expression was verified by immunoblot using anti-V5 antibody or anti-FLAG antibody, respectively, with antibodies to GAPDH providing a loading control. The mature MICB protein of 67 kDA is designated M whereas I refers to immature MICB retained in the ER by Rh159. Results shown are representative of two experiments. <b>C)</b> UL148 does not affect cell surface expression of NKG2DL. U373-NKG2DL cells were transduced with AdUL148 (blue) or AdCtrl (black) (MOI = 80) and, at 48 h, cells were harvested and analyzed for NKG2DL and TfR surface expression compared to isotype controls (dotted) by flow-cytometry. <b>D)</b> UL148 expression in C) was verified by immunoblot in each of the samples using anti-V5 antibody. GAPDH served as a loading control.</p

MICB is retained in the ER and associates with Rh159 in RhCMV infected cells.

<p><b>A)</b> Immature MICB is enriched in RhCMV-infected cells. U373-MICB cells were infected with RhCMV (MOI = 3) for 12 or 24 h or left uninfected prior to lysis in 1% NP40. Cell lysates were treated with Endoglycosidase H (E), PNGase F (P), or digestion buffer alone (-), separated by SDS-PAGE and immunoblotted with monoclonal antibodies (mAbs) to MICB or GAPDH. Mature glycosylated MICB (67kDa) is EndoH resistant (R), whereas immature MICB (I) remains EndoH sensitive with an apparent MW of 43kDa upon EndoH treatment (S) indicative of ER retention. <b>B)</b> RhCMV inhibits intracellular transport of MICB. U373-MICBs were infected with RhCMV (WT) (MOI = 3) for 24 h (visualization by light microscopy confirmed 100% cytopathic effect (CPE)) or left uninfected (NI) followed by metabolically labeling with [35S]cysteine and [35S]methionine for 30 min. The label was then chased for the indicated times (h), cells were lysed and MICB was immunoprecipitated from cell lysates using a MICB specific antibody. Precipitates were either digested with EndoH (+) or mock treated (-) followed by SDS-PAGE and autoradiography. Stars (*) indicate an EndoH-sensitive protein co-precipitating with MICB in infected cells. <b>C-D)</b> Isolation and identification of Rh159 co-immunoprecipitating with MICB. U373-MICB cells were infected with RhCMV (WT) or left non-infected (NI) as above and cells were lysed at 48 hpi. MICB was immunoprecipitated with anti–MICB mAb, the immunoprecipitates were separated by SDS-PAGE, and proteins visualized by Coomassie Blue staining. The IgG heavy chain (55kDa) is indicated. The 43kDa protein (*) was excised from the gel and digested with trypsin. <b>D)</b> Mass-spectrometric analysis by LC-MS/MS identified tryptic peptides corresponding to Rh159 (gray shaded boxes). The predicted amino acid sequence of Rh159 contains a signal sequence (italics), N-linked glycosylation sites (underlined), a C-terminal transmembrane anchor (bold), and an RXR ER retrieval motif (red). The results shown in A and B are representative of two or more independent experiments.</p