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CORD-19:45e64c5963b9f16e654cb31ff11561e1a9a420c3 JSONTXT 11 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function

Id Subject Object Predicate Lexical cue
T1 0-64 Sentence denotes Interactions of LSECtin and DC-SIGN/DC-SIGNR with viral ligands:
T2 65-127 Sentence denotes Differential pH dependence, internalization and virion binding
T3 129-137 Sentence denotes Abstract
T4 138-280 Sentence denotes The calcium-dependent lectins DC-SIGN and DC-SIGNR (collectively termed DC-SIGN/R) bind to high-mannose carbohydrates on a variety of viruses.
T5 281-418 Sentence denotes In contrast, the related lectin LSECtin does not recognize mannose-rich glycans and interacts with a more restricted spectrum of viruses.
T6 419-501 Sentence denotes Here, we analyzed whether these lectins differ in their mode of ligand engagement.
T7 502-704 Sentence denotes LSECtin and DC-SIGNR, which we found to be co-expressed by liver, lymph node and bone marrow sinusoidal endothelial cells, bound to soluble Ebola virus glycoprotein (EBOV-GP) with comparable affinities.
T8 705-854 Sentence denotes Similarly, LSECtin, DC-SIGN and the Langerhans cell-specific lectin Langerin readily bound to soluble human immunodeficiency virus type-1 (HIV-1) GP.
T9 855-1006 Sentence denotes However, only DC-SIGN captured HIV-1 particles, indicating that binding to soluble GP is not necessarily predictive of binding to virion-associated GP.
T10 1007-1153 Sentence denotes Capture of EBOV-GP by LSECtin triggered ligand internalization, suggesting that LSECtin like DC-SIGN might function as an antigen uptake receptor.
T11 1154-1226 Sentence denotes However, the intracellular fate of lectin-ligand complexes might differ.
T12 1227-1417 Sentence denotes Thus, exposure to low-pH medium, which mimics the acidic luminal environment in endosomes/lysosomes, released ligand bound to DC-SIGN/R but had no effect on LSECtin interactions with ligand.
T13 1418-1574 Sentence denotes Our results reveal important differences between pathogen capture by DC-SIGN/R and LSECtin and hint towards different biological functions of these lectins.
T14 1576-1638 Sentence denotes Differential pH dependence, internalization and virion binding
T15 1639-1865 Sentence denotes The lectin DC-SIGN (CD209) and the related lectin DC-SIGNR (L-SIGN, CD209L) bind to a virtually identical spectrum of viral and non-viral pathogens in a carbohydrate-dependent manner (Cambi et al., 2005; Koppel et al., 2005) .
T16 1866-1916 Sentence denotes Both lectins recognize high-mannose carbohydrates.
T17 1917-2069 Sentence denotes In addition, DC-SIGN binds to Lewis X sugars Feinberg et al., 2001; Guo et al., 2004; Lin et al., 2003; van Die et al., 2003; Van Liempt et al., 2004) .
T18 2070-2620 Sentence denotes DC-SIGN is expressed by dendritic cells (DCs) (Geijtenbeek et al., 2000) , platelets (Boukour et al., 2006; Chaipan et al., 2006) , activated primary B-cells (He et al., 2006; Rappocciolo et al., 2006) and certain macrophages (Soilleux et al., 2001 (Soilleux et al., , 2002 , whereas DC-SIGNR is found on vascular Available online at www.sciencedirect.com Virology 373 (2008) 189 -201 www.elsevier.com/locate/yviro endothelium in the placenta and on liver and lymph node sinusoidal endothelial cells (Bashirova et al., 2001; Pöhlmann et al., 2001c) .
T19 2621-2760 Sentence denotes In liver , lymph node and placenta (Soilleux et al., 2001 (Soilleux et al., , 2002 Pöhlmann et al., 2001c) , both lectins are co-expressed.
T20 2761-2989 Sentence denotes DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can concentrate virions on the cell surface, thereby facilitating engagement of the cognate cellular receptors and subsequent infectious viral entry into target cells.
T21 2990-3192 Sentence denotes DC-SIGN-mediated capture of human immunodeficiency virus type-1 (HIV-1) by DCs in the anogenital mucosa was roposed to facilitate dissemination of sexually transmitted HIV-1 (Geijtenbeek et al., 2000) .
T22 3193-3362 Sentence denotes However, several reports indicate that the contribution of DC-SIGN to augmentation of HIV-1 infectivity by DCs is minor (Boggiano et al., 2007; Gummuluru et al., 2003) .
T23 3363-3561 Sentence denotes Nevertheless, DC-SIGN on platelets or activated Bcells might promote HIV-1 spread once the virus has reached the blood stream (Boukour et al., 2006; Chaipan et al., 2006; Rappocciolo et al., 2006) .
T24 3562-3685 Sentence denotes DC-SIGNR in lymph nodes and liver might also impact spread of HIV-1 and hepatotropic viruses, like hepatitis C virus (HCV).
T25 3686-3964 Sentence denotes Indeed, polymorphisms in the DC-SIGNR neck region modulate susceptibility to HIV-1 infection (Liu et al., 2006) , and maybe more impressively, a correlation between the DC-SIGNR genotype and the viral load in HCV infected patients has been documented (Nattermann et al., 2006) .
T26 3965-4393 Sentence denotes While the former finding is controversial (Lichterfeld et al., 2003) , the observations that liver sinusoidal endothelial cells (LSEC) are permissive for HIV-1 infection (Steffan et al., 1992) and capture HCV envelope protein E2 in a DC-SIGNR-dependent manner Ludwig et al., 2004; Pöhlmann et al., 2003) indicate that DC-SIGNR and possibly other lectins on LSECs might impact spread of several clinically relevant human viruses.
T27 4394-4520 Sentence denotes The DC-SIGN/R and CD23 genes are located in a cluster of lectin encoding genes on chromosome 19p13.3 (Soilleux et al., 2000) .
T28 4521-4660 Sentence denotes Liu and colleagues (2004) reported that the gene for LSECtin, another calcium-dependent (C-type) lectin, is also localized in this cluster.
T29 4661-4797 Sentence denotes They found that LSECtin is co-expressed with DC-SIGNR in liver and lymph node and, like DC-SIGN/R, binds to mannose (Liu et al., 2004) .
T30 4798-4937 Sentence denotes We previously discovered that LSECtin, like DC-SIGN/R, augments Ebola virus (EBOV) and SARS coronavirus infection (Gramberg et al., 2005) .
T31 4938-5133 Sentence denotes However, in contrast to DC-SIGN/R, LSECtin failed to augment HCV and HIV-1 infection and virus interactions with LSECtin were not inhibited by the mannose polymer mannan (Gramberg et al., 2005) .
T32 5134-5315 Sentence denotes Thus, LSECtin might have an expression pattern similar to DC-SIGNR but it seems to interact with a more restricted spectrum of viruses and seemingly in a mannose-independent manner.
T33 5316-5590 Sentence denotes Interestingly, a recent study confirmed interactions between LSECtin and EBOV and detected LSECtin on dendritic cells and macrophages under certain conditions (Dominguez-Soto et al., 2007) , suggesting some similarities between the expression pattern of LSECtin and DC-SIGN.
T34 5591-5697 Sentence denotes However, both the natural function and the role of LSECtin in virus infection are incompletely understood.
T35 5698-5884 Sentence denotes Here, we sought to elucidate similarities and differences between DC-SIGN/R and LSECtin interactions with ligands in order to better understand the biological functions of these lectins.
T36 5885-6036 Sentence denotes We confirmed co-expression of DC-SIGNR and LSECtin on LSECs and found that both lectins bind to soluble EBOV glycoprotein (GP) with similar affinities.
T37 6037-6196 Sentence denotes LSECtin also bound to HIV-1-GP but failed to capture HIV-1 particles, demonstrating that capture of soluble GP is not necessarily predictive of virion capture.
T38 6197-6258 Sentence denotes Finally, LSECtin like DC-SIGN was able to internalize ligand.
T39 6259-6432 Sentence denotes However, ligand bound to LSECtin could not be dissociated by endosomal low pH, indicating that the intracellular fate of internalized LSECtin and DC-SIGN might be different.
T40 6433-6702 Sentence denotes In order to investigate LSECtin expression and function, we generated LSECtin-specific monoclonal antibodies by immunization of mice with bacterially expressed LSECtin, followed by generation of clonal B-cell hybridomas as described previously (Baribaud et al., 2001) .
T41 6703-6842 Sentence denotes Screening of hybridoma supernatants by ELISA identified four supernatants that specifically reacted with the antigen used for immunization.
T42 6843-6901 Sentence denotes Antibodies from the respective supernatants were purified.
T43 6902-7081 Sentence denotes All antibodies were reactive against recombinant LSECtin as assessed by ELISA and allowed detection of both recombinant and cellular LSECtin by Western blot (Table 1 ; Fig. 1A ).
T44 7082-7256 Sentence denotes However, only the D18 antibody was reactive against native, cell surface expressed LSECtin as judged by FACS analysis (Fig. 1A) and was therefore chosen for further analysis.
T45 7257-7376 Sentence denotes LSECtin is a type II transmembrane protein, which, based on sequence analysis, exhibits the following domain structure:
T46 7377-7499 Sentence denotes Nterminal cytoplasmic domain (CD), transmembrane domain, neck domain and C-terminal carbohydrate recognition domain (CRD).
T47 7500-7693 Sentence denotes In order to identify the LSECtin domain recognized by D18, various portions of LSECtin were bacterially produced (Fig. 1B) and D18 reactivity against the recombinant proteins assessed by ELISA.
T48 7694-7871 Sentence denotes D18 specifically reacted with the purified neck domain and all proteins containing this domain (Fig. 1C) , indicating that the D18 epitope is located in the LSECtin neck region.
T49 7872-8060 Sentence denotes DC-SIGNR and LSECtin are co-expressed in liver, lymph node and bone marrow, but only DC-SIGNR is found in placental tissue We next employed D18 to investigate tissue expression of LSECtin.
T50 8061-8209 Sentence denotes Immunohistochemical staining of parental 293 cells and cells engineered to express LSECtin confirmed the specificity of the antibody ( Fig. 2A, B) .
T51 8210-8542 Sentence denotes Staining of tissue sections revealed no evidence for LSECtin expression in any major organ/tissue, except lymph node (Fig. 2C, D) , liver (Fig. 2G ) and bone marrow (Fig. 2J) , where LSECtin was detected with a 2E , H, K) and was mainly due to DC-SIGNR expression (Bashirova et al., 2001; Lai et al., 2006; Pöhlmann et al., 2001c) .
T52 8543-8703 Sentence denotes Sinusoidal endothelial cells in liver (Fig. 2I ), but not lymph node (Fig. 2F) , also expressed CLEC-2, a novel HIV-1 attachment factor (Chaipan et al., 2006) .
T53 8704-9008 Sentence denotes In the liver, anti-LSECtin immunostaining was more similar to anti-CLEC-2 immunostaining, in keeping with LSECtin expression by sinus endothelial cells, rather than sinus endothelial cells and Kupffer cells, which have been reported to express both DC-SIGN and DC-SIGNR, and DC-SIGN alone, respectively .
T54 9009-9137 Sentence denotes Finally, DC-SIGNR (Fig. 2N ), but not LSECtin (Fig. 2M ) or CLEC-2 ( Fig. 2O) , was expressed by vascular cells in the placenta.
T55 9138-9350 Sentence denotes Thus, in agreement with previous results (Liu et al., 2004) , LSECtin shows a restricted tissue expression and is only found in liver, lymph node and bone marrow sinusoids, where it is co-expressed with DC-SIGNR.
T56 9351-9621 Sentence denotes In light of recent published data suggesting expression of LSECtin by human peripheral blood and thymic dendritic cells isolated ex vivo LSECtin and the Langerhans cell-specific lectin Langerin bind to soluble HIV-1 envelope protein but do not capture and transmit HIV-1
T57 9622-9718 Sentence denotes Our previous analyses failed to detect LSECtin interactions with HIV-1 (Gramberg et al., 2005) .
T58 9719-9910 Sentence denotes It was unclear, however, whether LSECtin simply failed to bind to the surface unit Gp120 of the HIV-1 envelope protein or whether the failure to capture HIV-1 was due to more complex reasons.
T59 9911-10070 Sentence denotes We therefore analyzed binding of a soluble HIV-1-Gp120-Fc fusion protein to CD4-negative HeLa cells transiently expressing DC-SIGN, LSECtin, Langerin and CD23.
T60 10071-10288 Sentence denotes Langerin, which is exclusively expressed by Langerhans cells (Valladeau et al., 1999) , was included as a positive control since we previously showed robust HIV-1-Gp120 binding to this lectin (Turville et al., 2002) .
T61 10289-10323 Sentence denotes CD23 served as a negative control.
T62 10324-10410 Sentence denotes Cell surface expression of all lectins was readily detectable (Fig. 3A , upper panel).
T63 10411-10562 Sentence denotes DC-SIGN and Langerin but not CD23 readily bound to HIV-1-Gp120 (Fig. 3A , lower panel), as expected (Geijtenbeek et al., 2000; Turville et al., 2002) .
T64 10563-10659 Sentence denotes To our surprise, LSECtin also captured HIV-1-Gp120 with high efficiency (Fig. 3A , lower panel).
T65 10660-10820 Sentence denotes The mannose polymer mannan blocked binding of HIV-1-Gp120 and, with slightly lower efficiency, binding of soluble EBOV-GP-Fc to DC-SIGN and Langerin (Fig. 3B ).
T66 10821-10997 Sentence denotes In contrast, mannan did not inhibit ligand binding to LSECtin (Fig. 3B) , providing further evidence that LSECtin does not recognize mannose residues on the surface of ligands.
T67 10998-11179 Sentence denotes Given the similar binding of soluble Env by DC-SIGN, LSECtin and Langerin, we next compared the ability of these lectins to support HIV-1 transfer to target cells (trans-infection).
T68 11180-11360 Sentence denotes While DC-SIGN-expressing cells captured and transmitted HIV-1 to target cells with high efficiency, LSECtin and Langerin-positive cells failed to bind and transmit HIV-1 (Fig. 4) .
T69 11361-11525 Sentence denotes Langerin expression also failed to enhance EBOV-GP-driven infection, while DC-SIGN and LSECtin augmented infection efficiency, as expected (Gramberg et al., 2005) .
T70 11526-11681 Sentence denotes Finally, all cell lines exhibited com-parable susceptibility to infection with a control virus bearing the G-protein of vesicular stomatitis virus (VSV-G).
T71 11682-11875 Sentence denotes Thus, despite efficient binding to HIV-1-Gp120, LSECtin and Langerin do not interact with HIV-1, at least under the conditions tested and thus may not support HIV-1 spread in infected patients.
T72 11876-11970 Sentence denotes Liver and lymph nodes are important early targets of EBOV infection (Geisbert et al., 2003a) .
T73 11971-12230 Sentence denotes In these tissues, LSECtin and DC-SIGNR are co-expressed on sinusoidal endothelial cells and both lectins are capable of augmenting infectious cellular entry of EBOV (Alvarez et al., 2002; Bashirova et al., 2001; Pöhlmann et al., 2001c; Simmons et al., 2003) .
T74 12231-12522 Sentence denotes Because DC-SIGN/R and LSECtin differ in their carbohydrate specificities (Gramberg et al., 2005) , we asked if these differences translate into different affinities for EBOV-GP, which might have important implications for EBOV attachment to liver and lymph node sinusoidal endothelial cells.
T75 12523-12783 Sentence denotes In order to assess affinities of LSECtin and DC-SIGNR for EBOV-GP, 293 cells expressing roughly comparable amounts of these lectins (Fig. 5A ) were incubated with purified soluble EBOV-GP-Fc fusion protein and binding was assessed by flow cytometry (Fig. 5B ).
T76 12784-12807 Sentence denotes Quantification Fig. 3 .
T77 12808-13035 Sentence denotes LSECtin binds to soluble HIV-1-Gp120 in a mannose independent fashion. (A) HeLa cells were transiently transfected with plasmids encoding the indicated lectins and lectin expression and HIV-1-Gp120 binding analyzed in parallel.
T78 13036-13237 Sentence denotes Lectin expression was determined by staining with an antibody directed against a C-terminal AU1 antigenic tag (DC-SIGN, Langerin, CD23) or with the LSECtin-specific monoclonal antibody D18 (top panel).
T79 13238-13476 Sentence denotes Lectin binding to an HIV-1-Gp120-Fc fusion protein was assessed by incubating lectin expressing cells with concentrated supernatant containing HIV-1-Gp120 followed by detection of bound protein with an Fc-specific antibody (bottom panel).
T80 13477-13584 Sentence denotes Similar results were obtained in two separate experiments. (B) Inhibition of HIV-1-Gp120 binding by mannan.
T81 13585-13773 Sentence denotes Binding of HIV-1-Gp120 to lectin expressing cells was analyzed as described for panel (A); however, cells were pre-incubated with PBS or mannan before addition of soluble envelope protein.
T82 13774-13853 Sentence denotes Binding to lectin expressing cells in the absence of inhibitor was set as 100%.
T83 13854-14177 Sentence denotes The results of a representative experiment are shown and were confirmed in two independent experiments. of binding efficiency and assessment of K D values employing a previously described method (Lozach et al., 2003) revealed comparable high affinity binding for LSECtin (K D = 21 nM) and DC-SIGNR (K D = 26 nM) (Fig. 5C ).
T84 14178-14338 Sentence denotes Thus, LSECtin and DC-SIGNR on liver and lymph node sinusoidal endothelial cells might be equally adept at capturing soluble EBOV-GP and possibly EBOV particles.
T85 14339-14548 Sentence denotes LSECtin internalizes soluble EBOV-GP and acidic pH does not dissolve the lectin-ligand complex DC-SIGN internalizes ligand for intracellular processing and subsequent MHC presentation (Engering et al., 2002) .
T86 14549-14624 Sentence denotes We investigated whether LSECtin was also capable of ligand internalization.
T87 14625-14772 Sentence denotes B-THP cells (Wu et al., 2004) expressing exogenous DC-SIGN or LSECtin, but not control B-THP cells, readily captured soluble Alexa-labeled EBOV-GP.
T88 14773-14908 Sentence denotes Binding was lectin dependent because a cell population double positive for lectin and EBOV-GP was readily detectable by flow cytometry.
T89 14909-15112 Sentence denotes The population of double positive B-THP LSECtin cells was relatively heterogeneous and less prominent than that observed with B-THP DC-SIGN cells due to reduced expression of LSECtin relative to DC-SIGN.
T90 15113-15511 Sentence denotes When cells were maintained at 4°C, EBOV-GP binding could be abrogated by trypsin digestion, which cleaved both DC-SIGN and LSECtin ( Fig. 6A and data not shown, the remaining LSECtin signal was most likely due to unspecific reactivity of B-THP LSECtin cells with antibodies; T.G. and S.P., unpublished observations), suggesting that EBOV-GP was localized to the cell surface under these conditions.
T91 15512-15709 Sentence denotes In contrast, shifting cells to 37°C for 5 or 15 min before trypsin digestion abrogated lectin expression but preserved the EBOV-GP signal, indicating that the soluble protein had been internalized.
T92 15710-15829 Sentence denotes After successful cellular uptake, the lectin-ligand complexes might be dissociated by low pH in endosomal compartments.
T93 15830-15920 Sentence denotes Alternatively, the complexes might remain stable at low pH and might traffic to lysosomes.
T94 15921-16080 Sentence denotes In order to distinguish between these possibilities, we assessed whether exposure to low pH released EBOV-GP from DC-SIGN/R and LSECtin expressing T-REx cells.
T95 16081-16185 Sentence denotes At neutral pH, all lectin expressing cells bound to EBOV-GP with comparable efficiency (data not shown).
T96 16186-16395 Sentence denotes Upon treatment with medium of pH 6.0 or lower, EBOV-GP was released from DC-SIGN/R, with the association of DC-SIGNR with ligand being slightly more resistant to low pH than DC-SIGNligand complexes (Fig. 6B ).
T97 16396-16563 Sentence denotes In contrast, even exposure to pH 5.5 did not dissolve LSECtin-EBOV-GP complexes (Fig. 6B) , suggesting that intact LSECtin-ligand complexes might traffic to lysosomes.
T98 16564-16673 Sentence denotes We generated and employed an LSECtin-specific monoclonal antibody to analyze LSECtin expression and function.
T99 16674-16871 Sentence denotes We show that LSECtin is co-expressed with DC-SIGNR on liver, lymph node and bone marrow sinusoidal endothelial cells but, unlike DC-SIGNR, is absent from vascular endothelial cells in the placenta.
T100 16872-16960 Sentence denotes LSECtin bound to soluble dimeric EBOV-GP and HIV-1-GP and enhanced EBOV-GP-driven entry.
T101 16961-17114 Sentence denotes However, LSECtin failed to capture and transmit HIV-1 to target cells, suggesting that LSECtin does not interact with trimeric virionassociated HIV-1-GP.
T102 17115-17310 Sentence denotes Binding of EBOV-GP to LSECtin triggered internalization and lectin-ligand complexes remained stable at low pH, indicating that intact LSECtin-ligand complexes might be transported into lysosomes.
T103 17311-17447 Sentence denotes Thus, LSECtin like DC-SIGN might function as an antigen uptake receptor, but the intracellular fate of their ligands might be different.
T104 17448-17629 Sentence denotes Notably, LSECtin has different specificities for virus interactions compared to DC-SIGN and whether it might promote cisor trans-infection of other viruses remains to be determined.
T105 17630-17722 Sentence denotes The LSECtin-specific antibody we have developed will be an important tool in these analyses.
T106 17723-17951 Sentence denotes This antibody recognized cell surface expressed LSECtin through an epitope present in the LSECtin neck region (Fig. 1) but did not appreciably diminish EBOV-GP interactions with LSECtin (data not shown), which occur via the CRD.
T107 17952-18173 Sentence denotes Staining of tissue sections revealed that LSECtin and DC-SIGNR are coexpressed by liver, lymph node and bone marrow sinusoidal endothelial cells (Fig. 2) , which is in agreement with a previous report (Liu et al., 2004) .
T108 18174-18293 Sentence denotes LSECs are exposed to blood from the systemic circulation and from the gut and exhibit powerful scavenger capabilities .
T109 18294-18404 Sentence denotes Lectins like mannose receptor (MR), DC-SIGNR and LSECtin might contribute to the scavenger function of LSECs .
T110 18405-18619 Sentence denotes Whether the interaction of these lectins with viruses present in the blood stream preferentially leads to viral uptake, processing and MHC presentation or augmentation of viral infectivity remains to be determined.
T111 18620-18885 Sentence denotes Notably, LSECs are permissive for HIV-1 infection (Wu et al., 2004) were incubated with Alexa647-labeled soluble EBOV-GP-Fc fusion protein on ice; subsequently, cells were kept on ice or shifted to 37°C for 5 min or 15 min to allow internalization of bound antigen.
T112 18886-18973 Sentence denotes After trypsin or control treatment lectin expression and EBOV-GP binding were analyzed.
T113 18974-19137 Sentence denotes The results of a representative experiment are shown and were confirmed in two separate experiments. (B) Lysosomal pH does not dissociate LSECtin-ligand complexes.
T114 19138-19388 Sentence denotes 293T-REx cells induced to express comparable amounts of the indicated lectins were incubated with soluble EBOV-GP-Fc fusion protein, unbound protein was washed away and the cells were treated for 20 min with media adjusted to the indicated pH values.
T115 19389-19433 Sentence denotes Bound protein was detected by FACS analysis.
T116 19434-19468 Sentence denotes Binding at pH 7.5 was set as 100%.
T117 19469-19713 Sentence denotes The results of a representative experiment are presented and were confirmed in three independent experiments. (Steffan et al., 1992) and polymorphisms in the DC-SIGNR neck region might impact susceptibility to HIV infection (Liu et al., 2006) .
T118 19714-19927 Sentence denotes Moreover, LSECs capture HCVenvelope protein via DC-SIGNR Ludwig et al., 2004) and certain variations in the DC-SIGNR neck region are associated with a reduced viral load in HCV patients (Nattermann et al., 2006) .
T119 19928-20057 Sentence denotes Thus, DC-SIGNR on LSECs and lymph node and bone marrow sinusoidal endothelium might promote HIV-1 and particularly HCV infection.
T120 20058-20269 Sentence denotes By contrast, LSECtin does not interact with these viruses but our cell culture data indicate that LSECtin could facilitate filovirus interactions with sinusoidal endothelium in liver, lymph node and bone marrow.
T121 20270-20419 Sentence denotes Potentially increased expression of LSECtin due to elevated cytokine production in the context of EBOV infection might facilitate these interactions.
T122 20420-20618 Sentence denotes Liver and particularly lymph nodes are prominent early targets of EBOV in experimentally infected macaques, whereas infection of bone marrow is observed at later stages (Geisbert et al., 2003a, b) .
T123 20619-20937 Sentence denotes Because infection of the vascular endothelium in these organs is only detected during the later stages of EBOV replication (Geisbert et al., 2003b) , one might postulate that LSECtin and DC-SIGNR mainly promote viral spread by virus capture and transmission to adjacent target cells, like hepatocytes or Kupffer cells.
T124 20938-21163 Sentence denotes In fact, LSECs can bind and transmit duck hepatitis B virus to adjacent hepatocytes but do not become infected themselves (Breiner et al., 2001) , suggesting that these cells, like DCs, might promote viral infection in trans.
T125 21164-21287 Sentence denotes LSECtin did not bind or transmit HIV-1 (Fig. 4) , which is in agreement with our previous results (Gramberg et al., 2005) .
T126 21288-21505 Sentence denotes However, LSECtin captured a soluble HIV-1 Gp120-Fc fusion protein with high efficiency (Fig. 3) , suggesting that the binding interface present in soluble Gp120 is obscured or absent in Env trimers on HIV-1 particles.
T127 21506-21565 Sentence denotes Notably, similar results were obtained with Langerin (Figs.
T128 21566-21745 Sentence denotes 3 and 4) , a C-type lectin specifically expressed on Langerhans cells (Valladeau et al., 1999) , probably the first cell type to come into contact with sexually transmitted HIV-1.
T129 21746-21925 Sentence denotes The discrepancy between Gp120 and virion binding by LSECtin indicates that soluble and virion-associated Gp120 might differ in terms of content and/or surface exposure of glycans.
T130 21926-22121 Sentence denotes Alternatively, the multimerization status of Gp120 (soluble Fc fusion protein is most likely dimeric while virion-associated protein is trimeric) or the lectin expression level might play a role.
T131 22122-22345 Sentence denotes For Langerin, the situation might be different because it has recently been proposed that Langerin efficiently captures HIV-1 and targets bound virions for degradation in intracellular compartments (de Witte et al., 2007) .
T132 22346-22474 Sentence denotes In light of these results, our finding that Langerin expressing HeLa cells did not capture HIV-1 can be explained in three ways.
T133 22475-22660 Sentence denotes Exogenous Langerin on HeLa cells, unlike endogenous Langerin on Langerhans cells, might not be capable of binding HIV-1, maybe because a cofactor present on Langerhans cells is missing.
T134 22661-22735 Sentence denotes Alternatively, as for LSECtin, lectin expression levels might play a role.
T135 22736-22871 Sentence denotes Thus, higher copy numbers of surface expressed Langerin might be required for binding to HIV-1 as compared to binding to soluble gp120.
T136 22872-23058 Sentence denotes In this context, it needs to be pointed out that Langerhans cells most likely express substantially higher levels of Langerin relative to the transfected cells used in the present study.
T137 23059-23145 Sentence denotes Finally, Langerin-bound HIV-1 might be rapidly endocytosed and degraded in HeLa cells.
T138 23146-23370 Sentence denotes However, the absence of any specific HIV-1 binding to Langerin expressing HeLa and 293T cells even early after exposure ( Fig. 4 and data not shown) suggests that HIV-1 degradation might not account for the effects observed.
T139 23371-23626 Sentence denotes In any case, it is notable that binding to soluble Gp120 is not necessarily predictive for binding to virus particles (present manuscript) and capture of virions does not necessarily result in transmission (Baribaud et al., 2001; Pöhlmann et al., 2001b) .
T140 23627-23792 Sentence denotes Detailed analysis of the determinants involved might yield important insights into the molecular mechanisms behind lectin-mediated augmentation of viral infectivity.
T141 23793-23926 Sentence denotes Ligand captured by DC-SIGN, but not DC-SIGNR , is endocytosed and transported into late endosomes/lysosomes (Engering et al., 2002) .
T142 23927-24078 Sentence denotes The acidic milieu in this compartment dissociates the lectin-ligand complex and DC-SIGN might be recycled to the cell surface (Engering et al., 2002) .
T143 24079-24278 Sentence denotes Soluble EBOV glycoprotein bound to LSECtin expressing cells at 37°C but not at 4°C was protected against protease digestion (Fig. 6A) , suggesting that LSECtin-ligand complexes were also endocytosed.
T144 24279-24494 Sentence denotes A recent study by Dominguez-Soto and colleagues (2007) confirms and extends this observation by showing that tyrosine-and diglutamic-containing motifs in the cytoplasmic domain mediate rapid LSECtin internalization.
T145 24495-24571 Sentence denotes LSECtin like DC-SIGN might therefore function as an antigen uptake receptor.
T146 24572-24636 Sentence denotes However, the fate of the endocytosed lectins might be different.
T147 24637-24808 Sentence denotes Low pH dissociated DC-SIGN/R-ligand complexes, whereas LSECtin-ligand complexes remained stable, even in an acidic milieu similar to that observed in lysosomes (Fig. 6B) .
T148 24809-24924 Sentence denotes Thus, both LSECtin and ligand might be degraded in lysosomes whereas DC-SIGN might be recycled to the cell surface.
T149 24925-25078 Sentence denotes Obviously, EBOV escapes degradation upon LSECtin-mediated uptake and uses this lectin for augmentation of infectivity ( Fig. 4 ; Gramberg et al., 2005) .
T150 25079-25401 Sentence denotes These results are not unexpected because endosomal/lysosomal enzymes were shown to dissociate GP1 (which is bound by LSECtin) from GP2 (which drives membrane fusion) (Chandran et al., 2005; Schornberg et al., 2006) , thereby most likely releasing EBOV from LSECtin and allowing the virus to fuse with the vesicle membrane.
T151 25402-25627 Sentence denotes In summary, LSECtin shows important similarities to DC-SIGN/R, including expression by liver, lymph node and bone marrow sinusoidal endothelial cells, interaction with viral glycoproteins and internalization of bound ligands.
T152 25628-25677 Sentence denotes However, important differences are also apparent.
T153 25678-25828 Sentence denotes For one, LSECtin promotes infection by a more restricted spectrum of viruses and might have no impact on HIV-1 and HCV spread in infected individuals.
T154 25829-26024 Sentence denotes Secondly, complexes between ligand and DC-SIGN/R but not LSECtin-ligand complexes are dissociated by a low pH milieu, suggesting differential intracellular trafficking of the endocytosed lectins.
T155 26025-26160 Sentence denotes 293T cells were propagated in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), penicillin and streptomycin.
T156 26161-26480 Sentence denotes 293T-REx cells expressing DC-SIGN, DC-SIGNR or LSECtin were described previously (Gramberg et al., 2005; Pöhlmann et al., 2001a,b; Simmons et al., 2003) and maintained in medium containing DMEM, 10% FBS, 50 μg/ml zeocin (Invitrogen, CA, USA), 2.5 μg/ml of blasticidin (Invitrogen, CA, USA), penicillin and streptomycin.
T157 26481-26599 Sentence denotes Expression was induced by culturing the cells in medium containing 0.1 μg/ ml of doxycycline (Sigma-Aldrich, Germany).
T158 26600-26721 Sentence denotes 293T-REx parental cells were maintained in the same medium as lectin expressing cell lines; however, no zeocin was added.
T159 26722-26846 Sentence denotes CEMx174 5.25 (Hsu et al., 2003) and HeLa cells were maintained in RPMI 1640 containing 10% FBS, penicillin and streptomycin.
T160 26847-27050 Sentence denotes Parental B-THP cells, which are derived from Raji-B-cells (Wu et al., 2004) , and B-THP cells expressing DC-SIGN and DC-SIGNR were maintained in RPMI 1640 containing 10% FBS, penicillin and streptomycin.
T161 27051-27238 Sentence denotes B-THP cells expressing LSECtin were generated via electroporation and FACS sorting and were maintained in RPMI 1640 supplemented with 10% FBS, penicillin, streptomycin and 500 μg/ml G418.
T162 27239-27281 Sentence denotes All cells were grown at 37°C and 5% CO 2 .
T163 27282-27409 Sentence denotes Expression plasmids encoding the indicated lectins were described previously (Pöhlmann et al., 2001a,b; Simmons et al., 2003) .
T164 27410-27535 Sentence denotes The glycoprotein (GP) expression plasmids employed for the generation of pseudotyped viruses were also described previously .
T165 27536-27719 Sentence denotes For regulated lectin expression, the open reading frames (ORFs) were inserted into pcDNA4TO (Invitrogen, CA, USA) as described previously (Gramberg et al., 2005; Marzi et al., 2004) .
T166 27720-28054 Sentence denotes For expression of soluble EBOV-GP and soluble HIV-1 Gp120, the extracellular portion of the respective GP-ORF was fused to the Fc part of human IgG 1 via PCR and cloned in frame with the aminoterminal murine IgG kappa signal peptide in the eukaryotic expression vector pAB61, as described (Gramberg et al., 2005; Marzi et al., 2004) .
T167 28055-28265 Sentence denotes For prokaryotic expression of LSECtin, PCR fragments spanning the entire LSECtin ORF or sequences encoding the indicated protein domains were inserted into pGEX6P1 (Amersham Biosciences, UK) via BamHI and XhoI.
T168 28266-28340 Sentence denotes All PCR-amplified sequences were confirmed by automated sequence analysis.
T169 28341-28468 Sentence denotes A mouse monoclonal antibody (MAb) directed against the AU1 antigenic tag was purchased from Covance Research Products, CA, USA.
T170 28469-28648 Sentence denotes The MAbs 507 (DC-SIGN specific), 526, DC28, DCN46 (DC-SIGN/R specific) and 604 (directed against DC-SIGNR) were obtained via the NIH AIDS Research and Reference Reagent Programme.
T171 28649-28814 Sentence denotes FITC-conjugated anti-mouse and Cy5-conjugated anti human secondary antibodies were purchased from Jackson ImmunoResearch, PA, USA, and Diaova, Germany, respectively.
T172 28815-29001 Sentence denotes The anti-LSECtin antibodies D18, C12, D9 and C17 were obtained by immunizing NMRI mice with 20 μg of purified GST-LSECtin fusion protein, as described elsewhere (Gramberg et al., 2005) .
T173 29002-29186 Sentence denotes Fusion proteins were obtained by overexpression in Escherichia coli DH10B fol-lowed by purification of the proteins employing Glutathione Sepharose 4B Beads (Amersham Biosciences, UK).
T174 29187-29289 Sentence denotes After a final boost, splenocytes were removed from immunized mice and fused with SP 2.0 myeloma cells.
T175 29290-29480 Sentence denotes Clonal hybridoma cells were cultured in HAT selection medium and screened for anti LSECtin reactivity via enzyme-linked immunosorbent assay (ELISA), Western blot analysis and flow cytometry.
T176 29481-29699 Sentence denotes Subsequently, antibodies were purified from supernatants reactive in Western blot (C12, D9, C17) or flow cytometry (D18) via HighTrap™ProteinG-Columns (Amersham Biosciences) according to the manufacturers instructions.
T177 29700-29899 Sentence denotes Soluble EBOV-GP-Fc and HIV-1-Gp120-Fc fusion proteins or control Fc were obtained by harvesting the supernatant of 293T cells 48 h after transient transfection with the respective expression vectors.
T178 29900-30031 Sentence denotes The supernatants containing the respective proteins were concentrated using Centricon Plus-20 centrifugal filters (Millipore, USA).
T179 30032-30196 Sentence denotes To measure lectin binding, we incubated comparable amounts of soluble protein, as judged by Western blot, with transiently transfected HeLa cells for 45 min on ice.
T180 30197-30368 Sentence denotes After washing with FACS buffer, cells were stained with Cy5-conjugated anti-human IgG (Jackson ImmunoResearch, USA) at a final concentration of 15 μg/ml for 45 min on ice.
T181 30369-30518 Sentence denotes Cells were then washed and diluted in FACS buffer and binding was measured via flow cytometry using a FACS-Calibur flow cytometer (Becton Dickinson).
T182 30519-30705 Sentence denotes To determine the pH dependence of GP binding, we inoculated lectin expressing 293 cells with the indicated soluble GPs and subsequently incubated for 20 min in ice-cold PBS (pH 5.0-7.5).
T183 30706-30805 Sentence denotes Staining with secondary antibody and detection via flow cytometry was performed as described above.
T184 30806-30935 Sentence denotes Soluble EBOV-GP-Fc was directly labeled with the Alexa647fluorochrome using the AlexaFluor647-labeling Kit (Invitrogen, CA, USA).
T185 30936-31088 Sentence denotes To assess internalization, we incubated lectin expressing B-THP cells with labeled protein on ice, washed and incubated at 37°C or on ice, respectively.
T186 31089-31229 Sentence denotes After fixation of the cells with 2% PFA (10 min/ice), surface molecules were cleaved off by treatment with 1mg/ml trypsin TPCK (Invitrogen).
T187 31230-31407 Sentence denotes Subsequently, cells were stained for lectin expression on ice (lectinspecific antibody, Cy3-labeled secondary antibody) and were analyzed via flow cytometry, as mentioned above.
T188 31408-31650 Sentence denotes HIV-1-derived pseudotypes were generated by co-transfection of 293T cells with GP expression plasmids coding for the indicated glycoproteins and pNL4-3 E − R − Luc plasmid, as described previously (Connor et al., 1995; Simmons et al., 2003) .
T189 31651-31818 Sentence denotes The production of replication competent HIV-1 NL4-3 reporter virus bearing the luciferase gene in place of nef has been described previously (Pöhlmann et al., 2001a) .
T190 31819-31956 Sentence denotes The culture supernatants were harvested 48 h after transfection, passed through 0.4 μm pore size filters, aliquoted and stored at − 80°C.
T191 31957-32254 Sentence denotes To assess lectin-mediated enhancement of infection, we seeded 293 cells or HeLa cells expressing the indicated lectins onto 96-well plates at a density of 1.0 × 10 4 cells per well and incubated with viral supernatants normalized for comparable luciferase activity upon infection of control cells.
T192 32255-32497 Sentence denotes Generally, the medium was replaced 12 h after infection and luciferase activities in culture lysates were determined 72 h after infection with a commercially available kit, following the recommendations of the manufacturer (Promega, WI, USA).
T193 32498-32722 Sentence denotes HeLa cells transiently expressing the indicated lectins were seeded onto 96-well plates and incubated with HIV-1 NL4-3 reporter virus normalized for capsid protein (p24) content by ELISA (Murex, Abbott Diagnostics, IL, USA).
T194 32723-32939 Sentence denotes After 3 h incubation at 37°C, the cells were washed three times with PBS and either lysed in 1% Triton X-100 followed by quantification of the amount of bound virus by p24-ELISA or cocultured with CEMx174 5.25 cells.
T195 32940-33038 Sentence denotes Luciferase activities in cell lysates were determined 72 h after cocultivation as described above.
T196 33039-33196 Sentence denotes To compare the affinities of LSECtin and DC-SIGNR for ligand, we determined the dissociation constant K D of both proteins for binding to soluble EBOV-GP-Fc.
T197 33197-33434 Sentence denotes To this end, we incubated 3 × 10 5 293T-Rex cells (Invitrogen, CA, USA) induced to express comparable amounts of DC-SIGNR and LSECtin with indicated amounts of soluble EBOV-GP-Fc (0-15 μg/ml) or control-Fc (0-15 μg/ml) for 45 min on ice.
T198 33435-33554 Sentence denotes Pilot experiments showed saturation binding upon incubation of cells with 15 μg/ml soluble EBOV-GP-Fc (data not shown).
T199 33555-33663 Sentence denotes After washing with FACS buffer, cells were stained with Cy5-conjugated secondary antibody for 45 min on ice.
T200 33664-33758 Sentence denotes Cells were then washed and diluted in FACS buffer and binding was measured via flow cytometry.
T201 33759-33858 Sentence denotes After subtracting unspecific binding, specific binding curves were fit in by non-linear regression.
T202 33859-34015 Sentence denotes K D values were calculated by the use of the Origin 6.0 software (Microcal Software, USA), following a previously described protocol (Lozach et al., 2003) .
T203 34016-34200 Sentence denotes To determine the reactivity and domain specificity of anti-LSECtin antibodies, we coated 1.5 μg/ml of the indicated GSTfusion proteins or GST-control protein onto 96-well ELISA plates.
T204 34201-34423 Sentence denotes After blocking free binding sites with blocking buffer (TBS buffer + 2% BSA), coated fusion proteins were incubated with 100 ng/μl of the indicated anti LSECtin antibodies or a control antibody for 2 h at room temperature.
T205 34424-34761 Sentence denotes After extensive washing, specific interactions of the LSECtin-MAbs with fusion proteins were assessed by measuring the reactivity of alkaline phosphatase bound to secondary antibody (Vector-Laboratories, USA) with 5 mM p-nitrophenylphosphate (Vector Labs, USA) in an ELISA plate reader (SpectraMAX 190, Molecular Devices, USA) at 405 nm.
T206 34762-34949 Sentence denotes Expression of LSECtin on 293T-REx cells was induced by doxycycline or PBS, the cells were pelleted, fixed in formalin, embedded in paraffin wax and immunostained as described for tissues.
T207 34950-35242 Sentence denotes Tissue microarrays (a generous gift from Professor Kevin Gatter, University of Oxford) and anonymised sections of a range of normal tissues were obtained from the Department of Cellular Pathology, John Radcliffe Hospital, Oxford, United Kingdom, with local Research Ethics Committee approval.
T208 35243-35567 Sentence denotes These samples were immunostained as described previously (Pöhlmann et al., 2001c) with mouse monoclonal anti-LSECtin or isotype-matched negative control, mouse MAb DC28, mouse MAb CD68 clone PGM1 (Dako) or goat polyclonal anti-CLEC-2 (R&D Systems) and detected using standard staining kits from Dako and Vector Laboratories.
T209 35568-35647 Sentence denotes All sections were carefully reviewed by an experienced histopathologist (E.S.).
T210 35648-35791 Sentence denotes Images were taken at room temperature with an Olympus BX40 microscope equipped with a 40/0.75 0.17 lens and a Nikon Coolpix 950 digital camera.
T211 35792-35853 Sentence denotes Images were analyzed with Adobe Photoshop version 7 software.