Schematic Representation of the SILAC Labeling and Purification Protocol

<p>HeLa cells were grown for 5 d in labeling medium to ensure complete labeling. They were then infected with either adenovirus vector (light sample) or adenovirus-expressing K5 (heavy sample). Then 24 h post-infection, cells were harvested and counted, and equal numbers of cells combined. Samples were lysed in a Dounce homogenizer and unlysed cells removed by centrifugation. Membrane and soluble proteins were separated by centrifugation. The membrane pellet was resuspended, and different membrane fractions were separated over a discontinuous sucrose gradient. The bands corresponding to the plasma membrane (PM), Golgi, and ER fractions were removed and the proteins pelleted. The resulting pellet was then washed with sodium carbonate to remove non-integral membrane proteins, followed by ammonium bicarbonate to remove salts and other impurities. The final pellet was resuspended in 8 M urea and digested with trypsin for MS/MS analysis.</p

Ubiquitination and Lysosomal Degradation of ALCAM in the Presence of K5

<div><p>(A) Ubiquitination of ALCAM was examined by co-transfection of HeLa cells with ALCAM-HA as well as the indicated E3 enzymes. Then 24 h post-transfection, cells were lysed in 1% CHAPS, and ALCAM-HA was immunoprecipitated using anti-HA antibody. Samples were resolved on an 8% SDS-PAGE gel, transferred to PVDF, and immunoblotted (WB) with the anti-ubiquitin (Ubi) antibody P4D1 (top) or anti-HA (bottom). Ubiquitinated ALCAM was visible upon co-transfection of K5 and MARCH-VIII, but not with the inactive K5DE12 mutant and the unrelated HIV immune modulator vpu.</p><p>(B) To determine whether ubiquitinated ALCAM was degraded by proteasomes or in lysosomes, the fate of newly synthesized ALCAM-HA was determined by metabolic labeling for 10 min with S³⁵ Met/Cys and chasing the label for the indicated times (hours) in the presence of the indicated inhibitors. Following lysis, ALCAM-HA was immunoprecipitated using the HA antibody and samples were treated overnight with endoglycosidase H followed by electrophoretic separation. Note the increased recovery of ALCAM at 8 h in the presence of the endosomal/lysosomal proton pump inhibitor concanamycin A (ConA), but not in the presence of the proteasomal inhibitor MG132 (50 μmol).</p><p>(C) Surface expression of ALCAM can be restored by overexpressing a dominant negative version of the AAA-ATPase Vps4, which is essential for targeting proteins to MVBs. HeLa cells were transfected as indicated together with GFP to identify transfected cells. Then 24 h post-transfection, cells were harvested and the surface expression of either MHC I or ALCAM was analyzed using flow cytometry. The graph shows the ratio of mean fluorescence intensity from K5-transfected cells to that of control cells after gating for GFP. Data are averaged from three separate experiments.</p></div

Protein Identification and Differential Peptide Quantification

<div><p>(A) Approximately 500–700 proteins were identified by at least one peptide in each fraction; however, fewer than 150 proteins per fraction were identified in all three replicates. Of these, only five proteins changed more than 1.5-fold in the plasma membrane (PM) fraction, while only one protein changed more than 1.5-fold in all three replicates of either the Golgi or ER fractions.</p><p>(B) Proteins present in all three replicates of each fraction were analyzed for their predicted subcellular distribution using annotations in the Swiss-Prot proteomics database.</p><p>(C) Comparison of signal intensities obtained for selected peptides from the indicated proteins. The red line indicates the actual raw data recovered from the mass spectrometer; the blue line is a hypothetical best fit line drawn to help analyze elution time of the light and heavy peptides. The orange arrows indicate the time at which the initial MS/MS scan was initiated. Peptides derived from unaffected proteins, such as CD44, display a similar intensity for both the light and heavy isotopes. Peptides derived from differentially expressed proteins, such as BST-2, show clearly different intensity peaks for the light and heavy peptides. Note the slightly different scale for intensity of each peptide.</p></div

K5 Downregulates ALCAM

<div><p>(A) C-terminally HA-tagged ALCAM was co-transfected with K5 or control plasmid. Then 24 h post-transfection, cells were harvested and the abundance of ALCAM-HA in each lysate was measured by Western blotting with anti-HA antibody.</p><p>(B) Cell surface expression of endogenous ALCAM in the presence or absence of K5 was determined by flow cytometry. HeLa cells were co-transfected with K5 and a green fluorescent protein (GFP)–expressing plasmid to identify transfectants. Then 24 h post-transfection, cells were harvested and stained with antibodies against MHC I, ALCAM, CD9, and CD29. Both vector (solid black) and K5-transfected (white) cells were gated for GFP-expressing cells.</p><p>(C) ALCAM downregulation by K5-related proteins was determined by transfecting HeLa cells with viral K3 family members (K3 and M153R) and the indicated human MARCH proteins. Also examined were mutant K5 proteins with enzymatically inactive RING-CH domains (K5-RING) or lacking acidic residues implicated in subcellular targeting (K5DE12). Neither K5 mutant reduced ALCAM levels. KSHV K3 was unable to downregulate ALCAM, whereas the myxomavirus M153R protein significantly reduced ALCAM surface expression. Two of the MARCH proteins, MARCH-IV and MARCH-IX, strongly downregulated ALCAM, while MARCH-VIII showed a minimal effect.</p><p>(D) To determine whether ALCAM expression was affected by KSHV, latently infected immortalized DMVECs were treated with PMA to induce expression of lytic genes including K5. Surface levels of either MHC I or ALCAM were measured by flow cytometry 24 and 48 h post-induction. CD81, measured at 24 h, was used as a control. The ratio between the mean fluorescence intensity of infected and uninfected samples from these experiments is shown as fold change on the right.</p></div

BST-2 Is Differentially Expressed in K5-Expressing Cells

<p>HeLa cells were transduced with Ad-WT, Ad-K5, Ad-vpu, or Ad-MARCH-VIII. Then 24 h post-infection, cells were harvested and whole cell lysates were analyzed for the abundance of BST-2 and MHC I by Western blotting. Note that BST-2 is highly glycosylated and runs as multiple bands. The high molecular weight band marked with an asterisk is non-specific. Equal protein loading was confirmed by visualizing the ER resident chaperone Bap31 as well as general protein staining with Ponceau red ([A], left). The intensity of each band was quantified using densitometry ([A], right). The specificity of BST-2 downregulation was confirmed by transfecting K5 or a catalytically inactive K5-RING mutant, which showed no effect on either MHC I or BST-2 (B). The protein bands corresponding to BST-2 are more intense in (B) than in (A) because of longer exposure of the autoradiograph as well as variation in electrophoretic separation. Note that the same lysates were analyzed in lanes 1–3 of the blots in both (A) and (B).</p

Virotherapy Using Myxoma Virus Prevents Lethal Graft-versus-Host Disease following Xeno-Transplantation with Primary Human Hematopoietic Stem Cells

<div><p>Graft-versus-host disease (GVHD) is a potentially lethal clinical complication arising from the transfer of alloreactive T lymphocytes into immunocompromised recipients. Despite conventional methods of T cell depletion, GVHD remains a major challenge in allogeneic hematopoietic cell transplant. Here, we demonstrate a novel method of preventing GVHD by <em>ex vivo</em> treatment of primary human hematopoietic cell sources with myxoma virus, a rabbit specific poxvirus currently under development for oncolytic virotherapy. This pretreatment dramatically increases post-transplant survival of immunocompromised mice injected with primary human bone marrow or peripheral blood cells and prevents the expansion of human CD3⁺ lymphocytes in major recipient organs. Similar viral treatment also prevents human-human mixed alloreactive T lymphocyte reactions <em>in vitro</em>. Our data suggest that <em>ex vivo</em> virotherapy with myxoma virus can be a simple and effective method for preventing GVHD following infusion of hematopoietic products containing alloreactive T lymphocytes such as: allogeneic hematopoietic stem and progenitor cells, donor leukocyte infusions and blood transfusions.</p> </div

MYXV-treatment prevents lethal GVHD in immunodeficient mice following infusion of primary human BM.

<p>NSG mice were sublethally irradiated and then transplanted with either PBS (mock, n = 5), 1×10⁷ primary human BM (n = 36) or 1×10⁷ primary human BM pre-treated with MYXV (n = 36). Mice were weighed twice per week to monitor body condition (<b>A</b>) and sacrificed either six weeks after transplant or when their body condition score measured 2 (<b>B</b>). Significant differences in survival were determined using the log-rank test (P<0.05). N.S.  =  not significant. Post-mortem, organs were extracted, fixed in formalin, sectioned and stained for the presence of infiltrating human CD3⁺ lymphocytes (<b>C</b>). Immunohistochemistry images shown are representative of results observed in five separate mice from the three engrafted cohorts stained for the presence of human CD3⁺ cells.</p

<i>Ex Vivo</i> MYXV treatment prevents GVHD while still preserving GVL.

<p>NSG mice either naïve or carrying preexisting U266 myeloma were sublethally irradiated and injected with 10×10⁶ human donor BM cells that had been either mock treated or treated with vMyx-GFP. Mice were monitored for signs of GVHD for six weeks after which BM was harvested from surviving mice and the levels of both pre-existing U266 myeloma load (<b>A</b>) and normal human HSC engraftment (<b>B</b>) were quantified using flow cytometry. Cells can be distinguished based on expression of HLA-A, B, C, HLA-A2.1 and CD45 (hematopoietic stem cell progeny are HLA-A, B, C⁺/HLA-A2.1⁻/CD45⁺ while U266 myeloma cells are HLA-A, B, C⁺/HLA-A2.1⁺/CD45⁻).</p

MYXV treatment prevents alloreactive human:human mixed lymphocyte expansion.

<p>To determine if MYXV inhibited expansion of allo-reactive human cells in HCT samples following human/human allo-stimulation in a mixed lymphocyte reaction assay, mock-treated or MYXV-treated human BM cells were incubated for 10 days with irradiated HLA-mismatched human feeder cells. Mock-treated BM stimulated with irradiated feeder cells showed significantly increased numbers of viable cells while MYXV-treated BM did not.</p

MYXV treatment prevents in vivo expansion of human donor T lymphocytes after transplant into immunodeficient mice while sparing engraftment of normal HSPCs.

<p>NSG mice were sublethally irradiated and transplanted with 1×10⁷ whole human BM cells. Six weeks after transplant, bone marrow from mice was harvested and analyzed for human hematopoietic engraftment (human CD45⁺/HLA-A, B, C⁺ double positive cells) by flow cytometry. Treatment with MYXV did not alter the proportion of animals with successful human hematopoietic engraftment (<b>A</b>) but did slightly reduce the total numbers of engrafted human cells in mouse bone marrow, which did not reach statistical significance (<b>B</b>). Irradiated mice transplanted with 1×10⁷ CD34-depleted BM (<b>C</b>) or 1×10⁶ human CD3⁺ cells (<b>D</b>) displayed lower overall levels of engraftment and this engraftment was significantly reduced by <i>ex vivo</i> MYXV-treatment. Irradiated mice transplanted with 1×10⁵ CD34⁺ selected human cells showed levels of human cell engraftment similar to those observed in mice transplanted with whole human BM. Levels of human cell engraftment were not affected by <i>ex vivo</i> MYXV-treatment (<b>E</b>). Significance was determined using Student's t-test (P<0.05). N.S  =  not significant. Lineage of engrafted cells was determined by staining HLA-A, B, C⁺ cells from murine BM with antibodies against human CD3, CD15, or CD19 (<b>F</b>).</p