Activating NK- receptors, homing selectins and inhibitory Siglecs recognize EBOLA-GP and HPV-L1NK

The Ebola virus glycoprotein (EBOV)-GP is extensively glycosylated. Its expression induces a physical alteration of surface adhesion molecules, which causes cell rounding and detachment of the infected cells. This phenomenon likely plays a crucial role in viral pathogenicity. In this study, we show that such morphological changes are cell line-dependent as well as dependent on the surface proteins that interact with EBOV-GP in cis and trans. We have generated data showing that natural killer (NK) cell receptors (NCRs: NKp44 and NKp46), selectins (CD62E/P/L) and inhibitory Siglecs function as receptors for Ebola-GP and human papilloma virus (HPV-L1). We used HEK293 cells transfected with Ebola-GP and recombinant fusion proteins containing the extracellular domain of each of these receptors linked to the Fc of human IgG1, which showed significant differences in their virus-binding behavior compared to HEK293 cells transfected with empty vector. Further, to demonstrate that EBOV-GP is a ligand for NKp44 and other NK-receptors, and to investigate their role in immune escape, we also used human HEK-293, HeLa- and hamster CHO-GP-transfectants. Our data show that the NK receptors NKp44 and NKp46 play a key role in recognizing EBOV (Ebolavirus) and strongly suggest that other inhibitory (Siglec-7, Siglec-5) and non-inhibitory homing receptors (P-Selectin, L-Selectin, E-Selectin, and DC-SIGNR/DC-SIGN) take part in the interaction with virus particles. In addition, we show that NKp44, and NKp46, Siglec-7, and -5, and P-, L-, E-selectins as well as of and DC-SIGNR/DC-SIGN bind to the artificial viral envelope of a lentiviral vector that contains EBOV-GP. Altogether we prove that NCRs and a range of other inhibitory and activating receptors can interact with viral envelope/capsid proteins and that such interaction could play an important role in the elimination of virus infected cells. Our findings could be used to develop new strategies for prevention and treatment of infections by these viruses. Author summary The innate immune system is able to recognize specifically certain virus components. Here we show that activating NK-cell receptors (NKp44, and NKp46) are involved in such interaction by using HEK293 and CHOK1 cells transfected with the Ebola virus glycoprotein (EBOV-GP) and by binding studies with purified EBOV-GP. In detail, we have found moderate to strong affinity of Siglecs (Siglec-7, and -5), selectins (P-, L-, E-Selectin) and DC-SIGNR/DC-SIGN to purified EBOV-GP, and to cells transfected with EBOV-GP as well as to the envelope of a lentiviral vector carrying the EBOV-GP. Our findings show that NKp44, and NKp46, Siglec-7, and -5, as well as P-and L-selectins have a strong affinity to EBOV-G.


Introduction
cells from different donors (30). Upregulation of CD69 on NK cells is closely linked with activation of several signal transduction pathways including survival and induction of cytokine production and cytolysis of targets (31,32). Fuller and colleagues demonstrated a correlation between increased expression of sialic acid on the surface of infected cells with resistance against activation of the complement system (30). EBOV infection is able to impair type-I IFN production by infected cells and block IFN response in uninfected cells (33,34). In addition, EBOV infection is able to induce massive NK cell apoptosis, thus avoiding NK function and impairing NK-mediated DC maturation (35)(36)(37)(38).
The killer cell immunoglobulin-like receptors (KIRs) as well as Siglecs, and CD94-NKG2A, are involved in the inhibitory signal cascade of NK cells. Most inhibitory receptors recognize specific MHC class I isoforms and thereby ensure tolerance of NK cells against self-antigens (39). NK-cell-activation involves KIR2DS1-5, NKG2D, CD16, and NCRs (natural cytotoxicity receptors NKp46, NKp44 and NKp30). Treatment with mAbs against NCRs results in a very effective block of NK cell activity and thus reduces NK cell cytotoxicity against certain tumor cells (40,41).
NKp46 (NCR1) and NKp44 are expressed by innate lymphoid cells (ILCs) of group 1 (ILC1), a subset of group three ILCs (NCR + ILC3) (42), and by γδ T cells (43). NKp46 is a 46 kDa type I transmembrane protein belonging to the immunoglobulin (Ig) superfamily, which is characterized by two extracellular C2-type Ig-like domains (D1 and D2) (40,41) (Fig. 1). The TM domain of NKp46 contains a positively charged arginine residue that mediates association with the negatively charged aspartate residue found in the TM domain of the ITAM signaling adaptors, CD3ζ or the Fc receptor common γ (FcRγ) (41) (Fig. 1). NKp44 (NCR2) is a 44 kDa protein, consisting of a single extracellular V-type Ig (IgV) domain (44)(45)(46), followed by a long stalk region and a hydrophobic transmembrane domain. The latter contains a charged lysine residue that leads to association with DAP12 adaptor with ITAM (47) (Fig. 1). Structural studies have shown that the IgV domain of NKp44 forms a saddle-shaped dimer with a positively charged groove on one side of the protein (47,48). The NKp44-1 isoform has potential inhibitory ITIM but also the capacity to perform an activating function via association with DAP12 (49).
NKp30 (NCR3) is a 30 kDa protein that, similarly to the NKp46, is expressed on all mature resting and activated NK cells as well as on ILC2 cells (50). It contains an IgV domain and a hydrophobic transmembrane domain with a charged arginine residue. This leads to its ability to associate with the ITAM adaptors, CD3ζ and/or FcRγ (51, 52) (Fig. 1). There are six alternatively spliced transcripts from the NKp30 gene, termed NKp30a-f. As a consequence, NKp30 and NKp46 surface expression on adaptive memory NK cells is reduced (53). Cellular ligands for NKp30 include nuclear factor BAT3 and the he B7 family B7-H6 (54,55).
In previous work, we were able to show that ligands for NKp30 and NKp44 can be detected on the surface and in intracellular [5] compartments of different tumor cells (56). These ligands often contain heparan sulfate linked to proteoglycans (57,58). Further we reported that NKp30 and NKp46 present on natural killer cells play a key role in the immune response in the presence of vaccinia and mouse pox virus (ECTV/ectromelia virus), as they bind to hemagglutinin (HA)-a component of the Vaccinia virus envelope (59). It should be noted that NKp30 triggered activation of NK cells is blocked by HA of Vaccinia virus, whereas NKp46 stimulates NK cells (59). Moreover, the pp65 matrix protein of human cytomegalovirus (HCMV) binds NKp30 and inhibits its function (60). The results indicate that NKp30 has a different role in NK-cell cytotoxicity (51,59,61). Further, interferon (IFN)-γ secretion by NK and non-NK cells plays a role in the antiviral effect (62,63). For example, type I interferons are essential for the activation of NK cells during vaccinia virus (VV) infection (64). A VV infection induces expression of ligands for the activating natural cytotoxicity receptors (NCRs), and increases the susceptibility of host cells to lysis by NK cells (65). In addition to VV, human immunodeficiency virus and herpes simplex virus also contribute to an upregulation of the expression of NCR ligands on infected cells (66,67).
NKp46 recognizes the sigma1 protein of reovirus (68). In addition, the HA proteins of sendai-, influenza-, and newcastle disease (ND) viruses are able to bind NKp46 and NKp44, and induce NK cell activation (69)(70)(71)(72). In the human influenza and ND viruses, this is achieved when HA on the virus capsid links to sialic acid residues located on the surface of target cells. For cellular uptake, another structural viral protein, termed neuraminidase, cleaves the glycosidic bond to sialic acid residues, and thus liberates the virus for fusion with the target cell membrane (59,69,(71)(72)(73). NKp44 interacts with envelope glycoproteins from the West Nile and dengue viruses E/M proteins (74).
DC-SIGN (CD209) and DC-SIGNR (DC-SIGN-related, CD299, CLEC4M) bind soluble ebola-glycoproteins (EBOV-GP) with similar affinity (75), as well as soluble human immunodeficiency virus type-1 (HIV-1) gp120. This interaction is inhibited in an environment with increased pH (75). Highly glycosylated gp120 on the HIV-1 envelope has strong binding avidity to tetramerized DC-SIGN clusters on the surface membrane of dendritic cells. DC-SIGN and DC-SIGNR are furthermore receptors for other virus proteins such as from Lassa and Hepatitis C viruses (76)(77)(78).
DC-SIGN and DC-SIGNR are calcium-dependent C-type lectins, which have high affinity for ICAM3 (CD50) (79). DC-SIGN has low affinity to weakly polysialylated NCAM-1 (80). Lysis of dendritic cells by NK cells with polysialylated CD56 dim is increased in the presence of an anti-DC-SIGN antibody, which inhibits the interaction between DC-SIGN and its ligands (e.g. NCAM-1) (80). Several unrelated viruses, including influenza virus, RSV and HIV can directly suppress NK cell activity. Binding to DC-SIGN can promote HIV and hepatitis C viruses to infect T-cells via dendritic cells (77, 81,82). HIV-infected lymphocytes [6] (viral core protein p24 positive) do not present detectable DC-SIGN ligands (ICAM-1, ICAM-3, DC-SIGNR) on their cell surfaces, but non-infected lymphocytes, carrying NK-specific marker NCAM1, present cell surface DC-SIGN ligands (ICAM-1, ICAM-3) (80). HIV infection causes leukocyte dysfunction e.g. by increasing the level of soluble adhesion molecules (ICAM-1, soluble L-and E-selectins) leading to altered cell interactions (83).
L-selectin is a type I transmembrane cell adhesion molecule expressed on most circulating cells, including neutrophils, dendritic cells, monocytes, B cells, NK and T cells. L-selectin is a major regulator for leukocytes during trans-endothelial migration. E-and P-selectin are expressed on endothelial cells at sites of inflammation, interact with receptors on the surfaces of leukocytes, while L-selectin is expressed on lymphocytes and binds to glycans on high endothelial venules of lymph nodes. The glycoprotein gp120 of HIV-1 binds L-selectin in solution and on the host cell membrane. Upon entry of HIV into CD4 + T cells, L-selectin is cleaved at the membrane proximal site by proteolysis, thus facilitating virus release from cells (84,85). L-selectin is clustered with Eselectin and CD34 in trans (86,87), whereby co-localization of L-selectin with ADAM17 is induced to the uropod (85).
An effective immune response must result from coordination between the activities of the humoral and cellular immune systems.
EBOV sGP and glycosylated-GP (GlycGP) modulate host immune responses (99). The EBOV-infection of DCs leads to deregulation of signal amplifications and polarization between DC and T-effector cells, which takes place between MHC-I and MHC-II peptide presentations and interaction with the TCRs on T-cells. These inappropriate DC/T-cell interactions can lead to apoptosis in T-cells, which will eliminate clonal expansion of CD4 helper T cells, block T-CD8 mediated cytotoxicity and antibody production by B cells. [7]

EBOV-GP binds NCRs, homing selectins and inhibitory Siglecs
We used purified EBOV-GP coated ELISA-plates to assess the binding to IgG1-Fc recombinant fusion proteins carrying NKp44-Fc, CD44-Fc, CD24-Fc, PSGL-1-Fc and the known Ebola receptors DC-SIGN/DC-SIGNR. Our results show differential binding of EBOLA-GP to these receptors. DC-SIGN (CD209) and its related C-type lectin DC-SIGNR (CD299) bind strongly to EBOV-GP, as previously shown (75). NKp44 binds moderately to EBOV-GP. NKp30-Fc, CD44-FC, PSLG-1-Fc, NKG2D-Fc and CD24 shed from placenta have no significant binding to the EBOV-GP (supplementary Fig1). We next extended our study to test the binding of NKp46-Fc, P-selectin-Fc, E-selectin-Fc, L-selectin-Fc and inhibitory Siglec7-Fc, Siglec5-Fc, Siglec3-Fc. In order to reduce non-specific binding of some fusion proteins with high binding capacity (e.g. P-selectin-Fc, Siglec5-Fc) we tested different protein-containing and protein-free blocking buffers. The results show that P-selectin-Fc, L-selectin-Fc (CD62P/L) and inhibitory receptor Siglec3, Siglec5-Fc, Siglec7, as well as DC-SIGN/DC-SIGNR bind to EBOV-GP to varying degrees (Fig. 2). Out of these, Siglec3-Fc and E-selectin-Fc showed relatively weak binding to EBOV-GP, while NKp30-Fc, PSGL-Fc, CD44-Fc, and CD24-Fc do not bind significantly to EBOV-GP, compared to the controls. These results were confirmed repeatedly under different conditions and with different batches of proteins and the binding tendencies were reproducible throughout all experiments ( Fig. 2).

Expression of EBOV-GP in HEK-239 and CHO-K1 cells
EBOV-GP is highly glycosylated and interacts with various surface proteins simultaneously, thus affecting the adhesive behavior of infected cells. For instance, after transfection with EBOV-GP, HeLa cells change their morphology and behavior to grow as non-adherent cells at a slow proliferation rate (data not shown). We have transfected CHOK1 and HEK-239 cells stably with different plasmids carrying the EBOV-GP gene. It is known that HEK-293 cells are particularly sensitive to the expression of EBOV-GP viral glycoprotein (22,24,100). In order to investigate the binding behavior of normally glycosylated EBOV-GP, HEK-293 cells were separately transfected with two different expression-vectors, pCAGGS.cm5 (+)/GP (Marburg) and pcDNA6 (+)/GP-7916-5 (Heidelberg). CHOK1 was transfected with the expression-vector pCDNA 3.1. Both plasmids (pcDNA6 and pCDNA 3.1) contain a CMV (cytomegalovirus)-promoter, while pCAGGS-EBOV-GP consists of a chicken b-actin / rabbit βglobin hybrid. These two different species [human (HeLa, HEK-293) and Chinese hamster ovary (CHO)] were used, as we aimed to investigate the different patterns of glycosylation and cis co-partners for GP at the cell surface of transfected HEK-293 cells in [8] human vs non-human cells and their respective effect on the interaction between EBOV-GP and the binding partners mentioned above. To determine GP-expression efficiency, we used a human monoclonal anti-ZEBOV GP antibody KZ52(101) with high affinity and a mouse monoclonal antibody (3B11) against EBOV-GP showing weak binding to GP-transfected HEK-293 cells.
The latter anti-GP antibody showed a trypsin-dependent binding of EBOV-GP in HEK-293-cells, while the human anti-GP did not show dependency on trypsin (Fig. 3). The binding of 3B11 antibody (102) was twofold increased after trypsin treatment of GP-transfected cells (Fig.3a left). This may be due to the fact that EBOV-GP is present on the surface of transfected human HEK-293 cells in a highly glycosylated manner, leading to interaction with neighboring surface adhesion-proteins in cis. Apparently, expression of the EBOV glycoprotein results in correct processing and surface expression. The glycosylation pattern of the protein on the cell surface is broadly similar to the glycoprotein of the viral envelope. However, the binding analysis of EBOV-GP transfected HEK 293 cells showed different binding patterns with the fusion proteins, pending on the level of GP expression (Fig.4c).
In addition, the fusion proteins have more than one cellular co-partner at the cell surface, which makes the comparison extremely difficult; hence, our strategy was to use a cell line from another species (e.g. CHOK1).

NCRs, homing selectins and inhibitory Siglecs bind to EBOV-GP transfected HEK-293 and CHO-K1 cells
Next, we used our fusion proteins (NCRs and the well-known Ebola-GP ligands CD209/CD299 (DC-SIGN/DC-SIGNR) for immunofluorescence staining of the human cell line HEK-293 EBOV-GP . We observed strong, moderate or relatively week but significant binding of the fusion proteins to HEK-293 EBOV-GP cells in comparison to control vector-transfected HEK-293 cells (Fig.4a).

Binding of the fusion proteins to EBOV-GP transfected CHO-K1 cells
HEK-293 cells are commonly used as host for the heterologous expression of membrane proteins because they have high transfection efficiencies, faithfully process translation and glycosylation of proteins, and have optimal cell size, morphology and division rate. In addition to the HEK-293 EBOV-GP cells, we used EBOV-GP transfected CHO-K1 cells to assess the binding behavior of various recombinant fusion proteins. HEK-293 and CHO share very similar protein modification machineries in the endoplasmic reticulum (ER) and Golgi (103). More importantly, CHO cells are able to produce complex types of recombinant proteins with human-compatible glycosylation. CHO cells, however, do not express Gal a2,6 ST, a1,3/4 fucosyltransferase or b-1,4-Nacetylglucosaminyl-transferase III (GnT-III), which are enzymes expressed in human cells (104)(105)(106). Our results show, that proteins of the selectin-family (P-, L-, E-selectin) bind strongly to EBOV-GP transfected CHO-K1 cells. Siglec-7, NKp44 and NKp46 also bind significantly better to the transfected than to control cells, while PSGL-1 Siglec-3, Siglec-5, DC-SIGN and DC-SIGNR did not show significant binding compared to control cells ( Fig 5). This is in agreement with the results of the ELISAassays shown in Fig. 2.
CD24/ CD44 and EBOV-GP function as binding co-partners for P / E / L-selectins in cis and trans. Cis interaction of CD24 / CD44 and EBOV-GP expressed on the cell surface with selectins P / E-is not stable, hence there is an exchange between binding partners pending on their glycosylation. It is known that CD44 is hardly detectable on HEK-293 cells (112,113). It should be considered that CD44 comprises a family originating from alternative splicing and undergoing posttranslational modifications [10] with diverse functions, such as cell adhesion on different cell types (113). Proteoglycan CD44V6 (homing receptor Pgp-1, a variant of CD44) interacts with integrins and has been linked to poor prognosis in several tumors, where it facilitates metastasis and homing into distant tissues (114). We hypothesized that the ligands for selectins (CD44, CD24) on the cell surface of HEK-293 EBOV-GP cells probably are more accessible than on the vector transfected HEK-293 cells. Indeed, our results demonstrate an increased binding of the anti-CD44 and anti-CD24 antibodies to EBOV-GP transfected cells compared to vector transfected HEK-293 control cells (Fig. 6a).
When we incubated EBOV-GP transfected HEK 293 cells with CD24-Fc / CD44-Fc chimeric soluble receptors, which bind P / E / L-selectins, or with P / E / L-selectin-Fc chimeric soluble receptors, which bind CD24 and CD44, the ligand-receptor interactions in cis were disrupted (Fig. 6a). These biophysical interactions may also explain the increasing availability of CD24 / CD44 to bind anti-CD44s, anti CD44v6 and anti-HLA abc on HEK-293 transfectants. The analysis showed that binding of NKG2D-FC to its cellular ligands MICA/B (MHC I chain related molecules) is reduced for both types of GP-transfected HEK-293 cells (see Fig. 6b), whereas CD24, CD44s and CD44v6 bindings are significantly increased. Furthermore, we observed that the binding of mAb versus DR3 was slightly increased, probably to support the apoptosis induction on effectors cells, while mAb against CD56 was not significantly changed. Our results with EBOV-GP transfected cells showed a reduction in MHC-I (Fig. 6a). In addition, we observed a lower binding of NKG2D-Fc chimeric soluble receptors to EBOV-GP transfected HEK 293 cells (Fig.6b), which indicates a reduction of its ligands MIC-A/B, as it has been shown before (115). Altogether, these data suggest that a by EBOV-GP reduced stimulation of NKG2D, together with the recruitment of Siglec-ligands on the surface of infected cells, may lead to a suppressive state of effector cells (Fig. 6b, bottom).

Binding of the chimeric soluble receptors to their ligands depends on sialic acid and heparan sulfate
P-and L-selectin recognize clustered O-sialoglycan-sulfated epitopes on glycans, for example on the proteoglycan CD44. Lselectin, like other selectins, recognizes sialylated Lewis x and sialylated Lewis a (sLea; Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAc) (87,116). All three selectins recognize sulfated and sialylated derivatives (116). In addition, L-selectin binds to O-glycosylated sialomucins (117). Many enveloped viruses, for instance influenza virus and Newcastle disease virus, bind to sialic acid residues located on the surface of target cells (72). For viral cell entry, a structural viral protein termed neuraminidase (F0) cleaves the glyosidic bond to sialic acid residues and thus unleashes the virus for fusion with the target cell membrane (59,69,(71)(72)(73)118). In order to analyze the involvement of sialic acid and heparan sulfate in the binding of the fusion proteins to EBOV-GP transfected [11] CHO-K1 cells, we treated the GP and mock transfected cells with heparanases I & III, and sialidase. Following treatment, we analyzed the impact of these enzymes on the binding of fusion proteins to EBOV-GP and mock transfected CHO-K1 cells (Fig. 7).
Treatment with heparanase resulted in a decreased binding of selectins to EBOV-GP transfected CHO-K1 cells. Sialidase treatment, on the other hand, showed a significantly higher binding of selectins to EBOV-GP.
The analysis of HEK-293 EBOV-GP cells showed inconsistent binding of the fusion proteins E-selectin-Fc and P-selectin-Fc, which could be explained by changes in the degree of sialylation and heparan sulfation of the cell surface proteins (fig 7). In agreement with this, we observed that different culture conditions of HEK-293 EBOV-GP cells changed the constellation and ratios of surface adhesion molecules and other glycoproteins, especially of selectin ligands (unpublished results and Fig. 4). CHO-K1 cells transfected with EBOV-GP, which were incubated with fusion proteins (NKp44-Fc, NKp46-Fc, and L / E / P-selectins-Fc) showed reduced binding following heparanase treatment, as was expected since it is known that heparan sulfate is the main ligand for NCRs (57,58). Binding of Siglec7 was not affected by heparanase, yet strongly sensitive to sialidase I/III treatment, which reduced its binding significantly, as can be explained by the fact that sialic acid is the main ligand of Siglecs.

Binding of fusion proteins to lentiviral particles displaying EBOV-GP
The GP glycoprotein serves the Ebola virus to infect host cells by binding to various docking proteins. After transduction, viruses can incorporate or co-package many host cell-derived non-viral surface proteins into their newly formed envelope (119)(120)(121).
The recombinant lentiviral particles were produced in HEK-293 cells stably transfected either with a plasmid expressing EBOV-GP (pcDNA6/V5-HisB-EBOV-GP) or just vector (pcDNA6/V5-HisB). After concentrating the lentiviral particles, they were used for coating ELISA-plates. The efficiency of coating was determined using anti-GP-hIgG-Fc and goat anti-hIgG-POX. The coated plates were incubated with the most relevant fusion proteins. The results are shown in Fig. 8. We found significant binding to lenti-EBOV-GP by NKp44, NKp46, CD62e/l/p (E-Selectin, L-Selectin, P-Selectin), inhibitory receptor siglecs (siglecs-5, siglecs-7), DC-SIGN, and DC-SIGNR. From these, Siglec-3 and -5 showed weaker binding. In contrast, NKp30, PSGL-1, CD44, CD24, syndecan-1 1 , and TIM-1 did not bind to the Lenti-EBOV-GP. The observed binding of DC-SIGN, DC-SIGNR and selectins to the lentiviral particles without EBOV-GP is in agreement with previous reports (75,77,81). The binding of TIM-1 to lentiviral particles irrespective of their content of EBOV-GP is due to the fact that TIM1 binds to cohesive phosphatidylserine, which originated from the cell surface of host HEK-293 cells transfected with vector or EBOV-GP, and were taken up by the virus envelope (120) (Fig. 8). Likewise, the binding of CD44 and PSGL1 fusion proteins to vector envelopes, independent of any GP presence, can be explained by their binding to cellular ligands from the host plasma membrane, which were taken up by the lentiviral envelopes. The above described interaction plays a role in the recognition of virus infected cells by immune effector cells, e.g. NK cells. Interestingly, besides these specific interaction partners, a second mechanism of interaction may occur. This is the interaction of other cellular targets, e.g. membrane-associated heparan sulfate proteoglycans, which are recognized by NKp30, NKp44 and NKp46 (123) from NK-cells.

HPV-L1 binds NCRs, Homing Selectins and inhibitory Siglecs
Human papillomavirus infection requires cell surface heparan sulfate. The first interaction of HPV virions with the epithelial cell surface is with heparan sulfate proteoglycans (HSPGs) as it was shown for HPV16 and HPV33 pseudovirions in COS-7 cells (124). Furthermore, expression of the integral membrane proteins syndecan-1 and syndecan-4 in K562 cells, which do not have common HSPGs, was shown to enhance the attachment of HPV16 L1 virus-like particles (VLPs) and HPV11 virions in good correlation with the expression levels of HSPGs. In contrast, GPI-anchored glypicans are not primary receptors for binding of HPV VLPs to keratinocytes (125). In order to further asses the binding of the fusion proteins to purified HPV-L1 virus-like particles coated on ELISA plates, we incubated them with the different fusion proteins. Our results suggest that especially NKp44, Pselectin, and L-selectin bind strongly to HPV-L1, while NKp46, Siglec-5 and Siglec-7 showed lower binding (Fig. 9). Out of the group of selectins, L-selectin showed the highest binding affinity to HPV-L1. Although we had HPV-L1-transfected HEK-293 cells, we could not use them to test binding of our fusion proteins, as HPV-L1 does not have a transmembrane domain and therefore is secreted.
1 Syndecan receptors are required for internalization of the HIV-1 tat protein. [13]

EBOV-GP protects HEK-293 cells from lysis by polyclonal NK cells
We have observed that GP expression by target cells reduced lysis caused by primary NK cells, although staining of MHC-I by W6/32 mAb was significantly reduced (Fig.6a). Thus, this unexpected reduction could result from masking HLA-I by EBOV-GP.
In this case, however, increased NK-cell activity could be anticipated. However, we reasoned that other mechanisms are responsible for the reduced killing of target cells by NK-cells. Therefore, we assessed the susceptibility of HEK-293 EBOV-GP cells to lysis by primary NK cells obtained from different donors in comparison to respective mock-transfected HEK-293 cells. As shown in antibodies directed toward NCRs reduce the activity of NK cells (Fig. 10a). Altogether, these results suggest a crucial role of EBOV-GP in mediating immune escape of transduced/infected cells. [14] Discussion NK cells are effector cells of the early innate immune response that play a critical role in the lysis of virus infected and tumor cells without requiring prior antigen stimulation (126,127). The functions of NK cells are regulated through a balance of activating and inhibitory signals, which are transmitted through particular receptor binding cytokines or ligand structures on interacting target cells and pathogens (52,(128)(129)(130)(131). Activating signals of NK cells lead to either exocytosis of cytotoxic granules that lyse the target cells (132)(133)(134)(135)  We decided to study the mechanism of this process and the limitations of the immune effector cells to eradicate Ebolavirus. It is our notion, that the elimination of virus infected target cells by NK-cells is based on the sum of their ligand-binding activities, which is determined by the integration of inhibitory (e.g. KIRs) and activating receptor signals. We and others have previously shown the interaction of NCRs with hemagglutinin-neuraminidase of Newcastle disease virus (NDV), poxviral hemagglutinin and influenza viruses (59,69,72). We hypothesized that activating NCRs play a key role in the recognition of structural glycoproteins of virtually any virus by NK effector cells and thus facilitate the elimination of pathogens. Therefore, we have investigated the binding of purified EBOV-GP and HPV L1 to a series of soluble ectodomains of NCRs, selectins, Siglecs and other homing receptor proteins fused to the Fc portion of human IgG1. Recently, it was reported that blocking Nkp30 by a specific antibody reduced lysis of Ebolavirus infected dendritic cells by NK effector cells (30). So far, it is unknown, to which degree the cell surface of virus-infected cells is changed by the presence of EBOV-GP and then can sterically hinder the interaction of host cell adhesion proteins with activating or inhibiting effector-cell receptors. Nevertheless, it is interesting that EBOV-GP expressing cells can directly switch off the function of NK and CD8 T cells (138)(139)(140). The mechanism by which EBOV-GP suppresses the activity of NK cells may simultaneously involve (i) the binding of different inhibitory Siglecs, (ii) a reduction in ligands of KIR-receptors (MHC-I), some of which have activating roles, such as those KIRs with short cytoplasmic domain associated with activating adaptors (e.g. 3DS1 and 2DS1-5) and (iii) a reduction of NKG2D ligands (MICA/B). Specifically, highly glycosylated viral EBOV-GP [15] protein leads to reduced expression or shedding of MHC-I in host cells, or acts in cis sterically so that interaction or polarization between virus-infected target and effector cells is blocked (22) (Fig 6a/b). Besides KIR family genes, NK cells encode a variety of other activating and inhibitory receptors. In addition, there is inter-individual genetic diversity that causes different levels and types of inhibitory (KIR) and activating receptors (NKG2D, NCRs) (59). Here, we observed a lower binding of NKG2D-Fc chimeric soluble receptors to EBOV-GP transfected HEK 293 cells corresponding to reduced expression of MICA/B (Fig. 6b, bot-tom), as has been reported before (115). Interestingly, we reported previously for Vaccinia virus (VV)-infected HeLa cells that the cumulative expression of NKG2D ligands (MICA, MICB, ULBP1, ULBP2, ULBP3, and ULBP4) was identical to that in noninfected cells, as detected with NKG2D-Fc (59,141,142). However, a detailed analysis of the NKG2D ligands revealed a differential modulation of single ligands (59). In contrast, infection of HeLa cells with the mouse poxvirus ECTV resulted in clearly reduced levels of the cumulative expression of NKG2D ligands, as detected by human NKG2D-Fc (59). These data suggest that a by EBOV-GP reduced stimulation of NKG2D, together with the recruitment of Siglec-ligands on the surface of infected cells, may lead to a suppressive state of effector cells.
Furthermore, our data suggest that EBOV-GP binds strongly or at least moderately to the chimeric soluble proteins L-and Pselectin, Siglec-7 and -5, NKp44, NKp46 and, to a lesser extent, to Siglec-3 (Figs. 2, 4, 5, 7, 8). DC-SIGN, DC-SIGNR, and TIM-1 are known cellular receptors for EBOV (88,90,(143)(144)(145). Additionally, we investigated binding of DC-SIGN-Fc, DC-SIGNR-Fc and TIM-1-Fc to HEK-293 EBOV-GP cells and to purified EBOV-GP coated on ELISA plates. Interestingly, we were able to demonstrate the binding of DC-SIGN and DC-SIGNR to purified EBOF-GP on ELISA plates, but not to HEK-293 EBOV-GP cells. A possible explanation is that EBOV-GP from the surface of transfected cells is fully occupied by interaction with its target proteins in cis, which causes that the binding sites for DC-SIGN-Fc and DC-SIGNR-Fc are not free for interacting in trans (Figs 2, 4, 5 and 8). In contrast, GP on the viral envelope remains free for interaction with both proteins. Like the HIV-1 gp120, the highly glycosylated EBOV-GP utilizes the C-type lectin receptor DC-SIGN (CD209) to infect dendritic cells, which are a major reservoir of EBOV (143)(144)(145). Soluble GP is post-translationally modified by N-glycosylation, but importantly also by C-mannosylation (9). DC-SIGNR specifically interacts with high-mannose N-linked carbohydrates on viral pathogens. Also, we could not prove binding of TIM-1-Fc to HEK-293 EBOV-GP cells or to purified EBOV-GP, yet we showed binding of TIM-1-Fc to lentiviral particles displaying EBOV-GP on their envelope (lenti-EBOV-GP), as well as to mock lentiviral particles. The binding of TIM-1-Fc to lentiviral particles irrespective of their content of EBOV-GP is due to the fact that TIM-1 binds to cohesive phosphatidylserine, which was incorporated by the virus envelope. It originates from the inner plasma membrane of mock or [16] EBOV-GP-transfected HEK-293 cells (120) (Fig. 8). TIM-3, TIM-4, and TIM-1 together inhibit HIV and Ebola virus release from infected cells (146). TIM-1 also serves as a pattern recognition receptor on invariant natural killer cells (iNKT), which mediate cell activation by TIMs binding to phosphatidylserine on the surface of cells undergoing apoptosis (147). It is very likely that TIM-3, an inhibitory checkpoint receptor on membrane effector cells, binds also to phosphatidylserine of the EBOV envelope (92,93). EBOV can induce dysfunction and even apoptosis in effector cells by binding to Siglecs-7,-5,-3,-8,-9, TIM-3 (91,(148)(149)(150)(151) and probably to other inhibitory receptors and thus cause immune escape (91,152,153). In line with this, a deficiency of immune effector cells has been observed during EBOV infection, whereby virus replication continues to be uncontrolled and renders immunotherapy of the disease difficult (154,155). The strength of NK cell cytotoxicity is controlled by an interplay of inhibitory (KIR, Siglecs, checkpoint proteins) and activating receptors (NCRs, NKG2D), as well as various costimulatory molecules (CD28, CD2) (156,157). A critical threshold of activation must be achieved for NK cells to mount a productive response (158,159). Viral infection can lead to immune suppression, either by downregulation of the cytotoxic function or by triggering apoptosis, leading to depletion of effector cells. Surprisingly, studies on EBOV-GP infected animals have shown a steady decrease in NK cell activity and the number of NK cells (140). It is unclear, to which extent adhesion molecules, which play important roles in interacting with the activating or inhibiting NK cell receptors, are affected by EBOV-GP in their relative expression, or through steric hindrance.
More in-depth knowledge about the mechanisms by which EBOV-GP expressing cells are able to directly switch off NK cell functions will certainly be helpful to develop an efficient therapy for EBOV infections (140).
Siglecs often function as sensor for sialylated glycoproteins. Through their intracellular ITIM, they induce strong inhibitory signaling upon binding to different linkages of sialic acid (160). Interestingly, this mechanism is used by tumor cells and pathogens to escape the immune system, by adding sialic acid residues to their glycan structures, thus highlighting that the sialic acid-Siglec interaction is key to the immune function against pathogens and cancer (161,162). NK and other effector cells express various Siglecs, e.g. Siglec-3, 7, 8, and 9 (163,164). Their presence stabilizes the conformation of membrane glycoproteins in cis interactions with endogenous sialo-conjugates at the cell surface of NK cells (164). Accordingly, we have observed binding of Siglec-7 and -5 to EBOV-GP in ELISA as well as to CHO-K1 EBOV-GP transfected cells and lentiviral particles. Siglec-7 binding to EBOBV-GP transfected CHO-K1 is dependent on 2,6 sialic acid linkage, since treatment with sialidase reverts the binding (Fig. 7). We have not tested Siglec-8 and -9, but strongly believe that Siglecs-8 and -9 also bind to GP because of their similar affinity for 2,6 and 2,3 sialic acid linkages. In addition, they are MHC class I-independent inhibitory receptors on immune effector cells, which -when stimulated-prevent the activation of these cells. However, the expression of the corresponding ligands [17] on target cells results in inhibition of NK cell-mediated cytotoxicity or apoptosis by interacting with Siglec-7 and -9 (164). The outermost position of sialic acids during the glycosylation process implies the capping of a variety of glycosylation structures (165). Indeed, sialic acid is increased on the surface of infected and tumor cells, which enables resistance against innate and adaptive immune responses and interference with the complement system (30).
Heparan sulfate binds many microorganisms and interacts with many viral envelope components e.g. HIV-1 (166), Hepatitis viruses (167,168), Flavioviruses (169,170), Vaccinia virus (171) All selectins (E-, L-and P-Selectin) bind specific fucosyl-sialyl-Lewis group carbohydrates of surface glycoproteins such as CD44v6, CD24, PSGL-1, and CD43 (87). CD44 is a receptor for hyaluronic acid, which is commonly expressed in hematopoietic cells, fibroblasts, and many tumor cells, which contributes to the adhesion of these cells to the extracellular matrix. Hyaluronic acid degradation fragments can disrupt the interaction between different CD44 variants and hyaluronic acid (176). Such disruption affects cell-cell and cell-matrix interactions, including cell traffic, immune cell homing and inflammation, cell aggregation, and release of chemokines or growth factors (177). In contrast to normal cells, tumor cells with upregulated CD44v6 are not able to bind hyaluronic acid. Unlike all other isoforms, CD44v6 is decorated by a fucosyl-sialyl-Lewis a group, which is transferred by fucosyltransferase 3 and this specific change blocks hyaluronic acid binding to CD44v6 (178,179). We have shown that L-, Pand E-selectins bind to purified EBOV-GP coated on ELISA plates and to EBOV-GP transfected cells (HEK-293 and CHO-K1). This is confirmed by recently published data that the viral particle gp120 of HIV-1 binds L-selectin (CD62L) (84)(85)(86)(87). Similarly, human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71 (180).
We conclude that selectins play an important role in virus release from infected cells, as it has been shown for HIV, which binds L-selectin and CD34 to co-localize with ADAM17 (84)(85)(86)(87). Glycans designated sialyl 6-sulfo Lewis x carbohydrate are potential ligands for selectins [15]. The interaction of the C-type CRD in the extracellular portion of each selectin with its receptor involves the direct binding of a fucose residue in the sialyl Lewis x tetrasaccharide to the conserved calcium ion characteristic of the C-type CRDs [11,12] The binding of NCRs to EBOV-GP as well as other viral envelope proteins, such as NDV, POX, HCMV, and Influenza, (59, 68-73, 181, 182) led us to examine their binding also to HPV-L1. Therefore, we tested the binding of the chimeric soluble proteins to [18] HPV L1 virus-like particles. NKp44, P-and L-selectins showed strong binding to HPV-L1, while NKp46, Siglec-5 and -7 showed low binding (Fig. 9). Out of the group of selectins, L-selectin was observed to have the highest binding affinity to HPV-L1. Based on our results it is conceivable, that NCRs will also bind to the spike of Corona virus, as was suggested recently 2 . Ovary ATCC ® CCL-61™) 3 . Cell lines were cultured in RPMI 1640 (Invitrogen, Karlsruhe, Germany) supplemented with 2 mM glutamine and 10% fetal calf serum (FCS). Human polyclonal NK cells were isolated by NK cell negative isolation kits (Invitrogen) from peripheral blood or from normal donor buffy coats obtained from the blood bank. Between 95 and 99% of NK cells were CD3 negative and CD56 positive. Cells were grown in Iscove's modified Dulbecco's medium (Invitrogen) with 10% human serum, penicillin-streptomycin, and 100 IU/ml IL-2 (NIH Cytokine Repository, Bethesda, MD).

Transfections
From the Institute of Virology in Marburg, Germany, we kindly received the vector pCAGGS-EBOV-GP for transfecting HEK-293-cells, which contains an AG promoter (chicken b-actin / rabbit b-globin hybrid). Furthermore, we used a blasticidin-expres-

ELISA-assay
For the direct detection of EBOV-GP via ELISA plates, we obtained the human Ebola Zaire (H. sapiens-wt) glycoprotein from Advanced Biomart (San Gabriel, CA, USA) and the anti-Ebola surface glycoprotein [KZ52] from Absolute Antibody (Oxford, GB). The obtained EBOV-GP was expressed with a polyhistidine-tag at the C-terminus and consists of 629 amino acids, predicting a molecular mass of 69.3 kDa. This antibody detects purified EBOV-GP coated on ELISA-plates and HEK-293 EBOV-GP cells by IF. MicroTest III enzyme-linked immunosorbent assay (ELISA) plates (BD Biosciences, Heidelberg, Germany) were coated overnight with EBOV-GP in 0.05 M NaHCO 3 -Na 2 CO 3 buffer (pH 9.6). They were also blocked using 3% skim milk powder (Merck, Darmstadt, Germany) in PBS-0.05% Tween 20 (Sigma-Aldrich) (PBS-T), as well as Pierce TM Protein-Free (PBS) Blocking Buffer (Thermo Scientific), overnight at 4° C. The recombinant fusion receptors listed above were obtained from R&D (Germany) and [20] analyzed regarding their binding ability to EBOV-GP coated ELISA-plates. All purified recombinant proteins (1 μg/100 μl in PBS-T with 1% bovine serum albumin) were added to EBOV-GP-coated wells at 2μg/well for 1 h at room temperature. After three times washing with PBS-T, peroxidase-conjugated goat anti-hIgG Fc or goat anti-mouse IgG Fc in PBS-T (1:2,000 with 1% bovine serum albumin) were added for 1 h at room temperature and then the plates were washed three times with PBS-T. Thereaf-  To concentrate the viral particles, they were centrifuged at 48,000×g for 3 h at 4°C, and viral pellets were re-suspended in cold PBS. Physical titration of the viral preparation was performed using p24 detection by sandwich ELISA. Microtiter plates precoated with anti-p24 Ab were incubated with increasing dilutions of the lentiviral suspension. After incubation and washing, p24 was quantified using a biotinylated anti-p24 Ab and detected using HRP-streptavidin. Color development was measured at 450 nm in a Bio-Rad spectrophotometer.          Treatment with heparanase resulted in decreased binding of selectins to EBOV-GP transfected cells. Sialidase treatment on the other hand caused a significantly increased binding of selectins to EBOV-GP. Binding of siglec7 was not affected by heparanase, yet strongly sensitive to sialidase I/III treatment, which reduced its binding significantly. This can be explained by the fact that sialic acid is the main ligand of siglecs. NKp44 and NKp30 have a reduced binding under heparanase treatment, as it is known [32] that heparan sulfate is the main ligand for NCRs. (7b) Control of the transfection efficiency with EBOV-GP and the control transfected with empty vector. The results show significant binding of NKP44-Fc, NKp46-Fc, E-selectin-Fc, L-selectin-Fc, E-selectins-Fc, and Siglec7-Fc. There was reduced binding of Siglec3-Fc and DC-SIGNR (CD299)-Fc to the GP containing vector lentiviral particle. There was equally increased binding of CD44-Fc PSGL-1-Fc and Tim-1 to the lentiviral vector with or without GP.