Tf2NH-Catalyzed Formal [3+2] Cycloaddition of Ynamides with Dioxazoles: A Metal-Free Approach to Polysubstituted 4-Aminooxazoles

An unprecedented Tf2NH-catalyzed formal [3 + 2] cycloaddition of ynamides with dioxazoles was developed to construct various polysubstituted 4-aminooxazoles. This approach features a metal-free catalytic bimolecular assembly of oxazole motifs; a low-cost catalyst, exceptionally mild reaction conditions, a very short reaction time, a broad substrate scope, and high efficiency. This metal-free protocol may find applications in pharmaceutical-oriented synthesis

Impaired proteolysis by SPPL2a causes CD74 fragment accumulation that can be recognized by anti‐CD74 autoantibodies in human ankylosing spondylitis

Ankylosing spondylitis (AS) is associated with autoantibody production to class II MHC-associated invariant chain peptide, CD74/CLIP. In this study, we considered that anti-CD74/CLIP autoantibodies present in sera from AS might recognize CD74 degradation products that accumulate upon deficiency of the enzyme signal peptide peptidase-like 2A (SPPL2a). We analyzed monocytes from healthy controls (n = 42), psoriatic arthritis (n = 25), rheumatoid arthritis (n = 16), and AS patients (n = 15) for SPPL2a enzyme activity and complemented the experiments using SPPL2a-sufficient and -deficient THP-1 cells. We found defects in SPPL2a function and CD74 processing in a subset of AS patients, which culminated in CD74 and HLA class II display at the cell surface. These findings were verified in SPPL2a-deficient THP-1 cells, which showed expedited expression of MHC class II, total CD74 and CD74 N-terminal degradation products at the plasma membrane upon receipt of an inflammatory trigger. Furthermore, we observed that IgG anti-CD74/CLIP autoantibodies recognize CD74 N-terminal degradation products that accumulate upon SPPL2a defect. In conclusion, reduced activity of SPPL2a protease in monocytes from AS predisposes to endosomal accumulation of CD74 and CD74 N-terminal fragments, which, upon IFN-γ-exposure, is deposited at the plasma membrane and can be recognized by anti-CD74/CLIP autoantibodies

Silencing the shutoff protein of Epstein-Barr virus in productively infected B cells points to (innate) targets for immune evasion

During productive infection with Epstein-Barr virus (EBV), a dramatic suppression of cellular protein expression is caused by the viral alkaline exonuclease BGLF5. Among the proteins downregulated by BGLF5 are multiple immune components. Here, we show that shutoff reduces expression of the innate EBV-sensing Toll-like receptor-2 and the lipid antigen-presenting CD1d molecule, thereby identifying these proteins as novel targets of BGLF5. To silence BGLF5 expression in B cells undergoing productive EBV infection, we employed an shRNA approach. Viral replication still occurred in these cells, albeit with reduced late gene expression. Surface levels of a group of proteins, including immunologically relevant molecules such as CD1d and HLA class I and class II, were only partly rescued by depletion of BGLF5, suggesting that additional viral gene products interfere with their expression. Our combined approach thus provides a means to unmask novel EBV (innate) immune evasion strategies that may operate in productively infected B cells

The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells

<div><p>Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.</p></div

EBV gp150 is broadly acting, but displays a degree of specificity for antigen-presenting molecules.

<p>Flow cytometric analyses of MJS-CD1d cells (adherent fraction) three days post transduction with gp150-IRES-GFP lentiviruses. A) Total EBV gp150 levels were determined by intracellular staining of permeabilized cells. Surface levels of HLA I, II, CD1d, CD10, and CD54 were determined using Ab staining on non-permeabilized cells. B) Surface levels of CD1d and CD54 are depicted as log MFI values with 95% confidence intervals, for GFP⁺ versus GFP^- MJS-CD1d cells transduced with gp150 or a GFP control. The slopes of the connecting lines reflect the declines in fluorescence (Δlog MFI) and, thus, the downregulation of HLA I, II, CD1d, CD10, and CD54 induced by gp150 (for the GFP control, the Δlog MFI did not significantly differ from 0). C) Cell surface levels of HLA I, II, CD10, CD34, CD44, and CD54 were determined using surface Ab staining. EBV gp150-induced downregulation of these molecules was calculated as in B) and represented as Δlog MFI.</p

Immune evasion by EBV gp150 occurs at the cell surface.

<p>A) The glycosylation status of CD1d and HLA I molecules in lysates of MJS-CD1d-gp150 and control cells was analysed by Western blot. To this end, denatured post-nuclear lysates of different FACS sorted cell populations (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005550#ppat.1005550.s004" target="_blank">S4 Fig</a>) were treated with Endo H or PNGase F to remove N-linked glycans. Endo H digestions served to examine protein transport beyond the cis-Golgi compartment; PNGase F digestions revealed the deglycosylated protein backbone (minus carbohydrates, -CHO). Actin was a loading control. B) Using the same experimental setup as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005550#ppat.1005550.g004" target="_blank">Fig 4C</a>, MJS-A2-GFP cells were transduced with a lentivirus encoding HA-gp150ΔC. Cell surface levels of CD1d, HLA I, and HLA-A2 (BB7.2) and total levels of HLA-A2 (GFP) were compared for (non-permeabilized) gp150^- and gp150⁺ cells. C) Confocal microscopy of non-permeabilized MJS-A2GFP-HA-gp150ΔC cells. Cell surface stains were performed for HLA-A2 (BB7.2) and gp150ΔC (HA). D) Quantification of surface expression of the indicated molecules based on multiple microscopy pictures.</p

gp150 interferes with surface display of Ag-presenting molecules during productive EBV infection.

<p>A) EBV⁺ AKBM-CD1d BL cells were treated for 20 hours with anti-human IgG Ab to induce viral replication. EBV-producing cells were identified by induced expression of the lytic cycle reporter rat CD2-GFP. In the presence of PAA, productive infection is arrested before late protein expression. Surface levels of the EBV late complex gHgL and of the Ag-presenting molecules HLA I, II, and CD1d were determined by flow cytometry. Histograms depict overlays to allow comparison of latently (GFP^-) and lytically (GFP⁺) infected B cells. B) 293T-CD1d cells were transfected with expression vectors encoding late EBV glycoproteins. Glycoproteins known to require a viral interaction partner were transfected together (BMRF2/BDLF2 and gM/gN). EBV protein expression was deduced from coexpression of GFP (BMRF2/BDLF2) or on the basis of a C-terminal tag (gM/gN, gB, gp350, gp150). Cell surface CD1d was stained prior to an intracellular staining for the tagged EBV proteins. Surface levels were compared between non-transfected and transfected cells. C) 293T-CD1d cells were transfected with N-terminally HA-tagged gp150 or an empty IRES-GFP vector control. Total gp150 expression was determined by intracellular staining and was compared to levels in EBV-producing B cells. The other histograms depict surface levels of HA (HA-gp150), HLA I, and CD1d staining for empty vector-transfected and gp150⁺GFP^high 293T-CD1d cells; GFP^high gating as indicated in the dot plots. D) Expression of gp150 was assessed at different time points during productive EBV infection of B cells; the early EBV antigen BGLF5 was taken along as a control (time points 6–10 hours and 12–20 hours are from different experiments).</p

The mechanism of gp150-induced immune evasion relies on sialoglycans shielding surface-exposed Ag-presenting molecules.

<p>A-D) CHO and Lec2 cells expressing human β₂m, HLA I, and CD1d were lentivirally transduced to co-express gp150 or HA-gp150ΔC and GFP (from EF1a and PGK promoters, respectively). A) Lectin (WGA-FITC) binding confirmed the glycosylation defect in Lec2 cells compared to parental CHO cells. B) Migration height of HA-gp150 was visualised by Western blot analysis. C-D) Levels of HLA I and CD1d at the surface of HA-gp150ΔC⁺GFP⁺ (gp150⁺) cells were compared to those on control, non-transduced GFP^- (gp150^-) cells. D) Surface levels of HLA I and CD1d are depicted as log MFI values with 95% confidence intervals, for gp150⁺ versus gp150^- wt CHO cells or sialylation-defective Lec2 cells. The slopes of the connecting lines (Δlog MFI) reflect the downregulation induced by gp150. Statistical analysis was performed using two-way ANOVAs and significance of the interaction term was assessed, as described in the Material and Methods section. One representative experiment of at least six is depicted. * p<0.01. E-L) MJS-CD1d-gp150 cells were generated by transduction with a lentivirus encoding both gp150 and GFP (from a CMV promoter and an IRES sequence, respectively) and were analysed 3 days post-transduction by Western blot and by flow cytometry, as for A-D with the modifications indicated below. E-H) To prevent sialylation, cells were treated with the sialic acid transferase inhibitor (500 μM inhibitor) fluorinated P-3F_ax-Neu5Ac. As a control, cells were treated with the non-fluorinated compound (500 μM, ctrl) for 4 days, starting 1 day prior to transduction. This control treatment was comparable to when cells were left untreated. E) Lectins SNAI, MALII, or PNA were used to detect sialoglycans or desialylated glycans, respectively. G) gp150⁺GFP^high cells were compared to non-transduced cells. H) One representative experiment of two is depicted. * p<0.01. I-L) To remove surface sialylation, intact cells were treated with neuraminidase (1U/μl, 60 min, 37°C) prior to cell lysis or lectin/Ab staining. L) One representative experiment of three is depicted. * p<0.01.</p

Glycan shielding of surface Ag-presenting molecules by gp150 occurs in human B cells and is reversed during productive EBV infection when gp150 is deleted.

<p>A-B) Latent AkataΔgp150 cells were lentivirally transduced to co-express either HA-gp150 or HA-gp150ΔC and GFP (from EF1a and PGK promoters, respectively) and were puromycin-selected to obtain pure populations of gp150⁺ cells. B cells already grow in suspension and gp150-positive B cells were maintained in culture for several weeks, indicating that gp150 expression was not toxic to the cells. A) Flow cytometry was used to assess total (intracellular staining with anti-gp150 Ab on permeabilized cells) and surface (anti-HA Ab on non-permeabilized cells) levels of EBVgp150. Expression levels of gp150 were compared to lytically induced AKBM cells (20 hours anti-human IgG treatment, rat CD2GFP⁺ cells). B) Akata+gp150 and non-transduced control cells were left untreated or were treated with neuraminidase (1U/μl, 60 min, 37°C). Surface levels of Ag-presenting molecules and cellular CD10 as a control were compared to gp150^- non-transduced cells. One representative experiment of three is depicted. C) Viral reactivation was induced in Akata wt and Δgp150 B cells by overnight culture with anti-human IgG and EBV-producing cells were identified by staining for the late viral protein gp350. Surface levels of Ag-presenting molecules and CD10 on lytic (gp350⁺) and latent (gp350^-) cells are depicted in overlay histograms. One representative experiment of at least four is depicted. Statistical analysis was performed for B and C) as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005550#ppat.1005550.g006" target="_blank">Fig 6</a>. * p<0.05, ** p<0.01.</p

Immune evasion by EBV gp150 does not depend on degradation of Ag-presenting molecules nor on gp150’s cytoplasmic tail.

<p>A) Schematic overview of the gp150 constructs and Abs used. Signal peptide, transmembrane domain, and cytoplasmic tail are abbreviated as signal pept, TM, and cytopl tail, respectively. B) Three days after lentiviral transduction, MJS-CD1d-gp150 cells were cultured overnight in the presence of an inhibitor of proteasomes (15nM epoxomicin) or of endolysosomal proteolysis (25μM chloroquine). Subsequently, flow cytometry was used to determine surface levels of HA-tagged gp150 (HA-gp150; detected with anti-HA Ab), HLA I, and mature CD1d (Ab #42) complexes on non-permeabilized cells and intracellular staining was performed to detect immature CD1d (Ab D5) molecules in permeabilized cells. C) MJS-GFPmycA2 cells were transduced with a lentivirus encoding HA-gp150 (upper) or HA-gp150ΔC (lower). Cell surface levels of CD1d, HLA I, and HLA-A2 (BB7.2 and anti-myc tag) and total HLA-A2 (GFP) levels were compared for gp150^- and gp150⁺ cells.</p