Interactions of LSECtin and DC-SIGN/DC-SIGNR with viral ligands: Differential pH dependence, internalization and virion binding.

The calcium-dependent lectins DC-SIGN and DC-SIGNR (collectively termed DC-SIGN/R) bind to high-mannose carbohydrates on a variety of viruses. In contrast, the related lectin LSECtin does not recognize mannose-rich glycans and interacts with a more restricted spectrum of viruses. Here, we analyzed whether these lectins differ in their mode of ligand engagement. 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. Similarly, LSECtin, DC-SIGN and the Langerhans cell-specific lectin Langerin readily bound to soluble human immunodeficiency virus type-1 (HIV-1) GP. However, only DC-SIGN captured HIV-1 particles, indicating that binding to soluble GP is not necessarily predictive of binding to virion-associated GP. Capture of EBOV-GP by LSECtin triggered ligand internalization, suggesting that LSECtin like DC-SIGN might function as an antigen uptake receptor. However, the intracellular fate of lectin-ligand complexes might differ. 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. Our results reveal important differences between pathogen capture by DC-SIGN/R and LSECtin and hint towards different biological functions of these lectins.

Modulation of virion incorporation of Ebolavirus glycoprotein: effects on attachment, cellular entry and neutralization.

The filoviruses Ebolavirus (EBOV) and Marburgvirus (MARV) cause severe hemorrhagic fever in humans and are potential agents of biological warfare. The envelope glycoprotein (GP) of filoviruses mediates viral entry into cells and is an attractive target for therapeutic intervention and vaccine design. Here, we asked if the efficiency of virion incorporation of EBOV-GP impacts attachment and entry into target cells and modulates susceptibility to neutralizing antibodies. In order to control the level of EBOV-GP expression, we generated cell lines expressing the GPs of the four known EBOV subspecies in an inducible fashion. Regulated expression of GP on the cell surface allowed production of reporter viruses harboring different amounts of GP. A pronounced reduction of virion incorporation of EBOV-GP had relatively little effect on virion infectivity, suggesting that only a few copies of GP might be sufficient for efficient engagement of cellular receptors. In contrast, optimal interactions with cellular attachment factors like the DC-SIGN protein required incorporation of high amounts of GP. Antibody-mediated neutralization of virions bearing high amounts of GP was slightly more efficient than neutralization of virions harboring low amounts of GP, suggesting that the efficiency of GP incorporation into virions might modulate susceptibility to neutralizing antibodies. Finally, regulated expression of GP in permissive 293 cells did not reduce EBOV-GP-driven infection but diminished vesicular stomatitis virus GP (VSV-G) and amphotropic murine leukemia virus (A-MLV) GP mediated entry in a dose-dependent manner. Therefore, intracellular GP does not seem to downmodulate expression of its receptor(s) but might alter expression and/or function of molecules involved in VSV-G and A-MLV-GP-dependent entry. Our results suggest that the efficiency of virion incorporation of GP could impact EBOV attachment to target cells and might modulate control of viral spread by the humoral immune response.

Evidence that multiple defects in murine DC-SIGN inhibit a functional interaction with pathogens.

Certain viruses, bacteria, fungi and parasites target dendritic cells through the interaction with the cellular attachment factor DC-SIGN, making this C-type lectin an attractive target for therapeutic intervention. Studies on DC-SIGN function would be greatly aided by the establishment of a mouse model, however, it is unclear if the murine (m) homologue of human (h) DC-SIGN also binds to pathogens. Here, we investigated the interaction of mDC-SIGN, also termed CIRE, with the Ebolavirus glycoprotein (EBOV-GP), a ligand of hDC-SIGN. We found that mDC-SIGN neither binds EBOV-GP nor enhances infection by reporterviruses pseudotyped with EBOV-GP. Analysis of chimeras between mDC-SIGN and hDC-SIGN provided evidence that determinants in the carbohydrate recognition domain and in the neck domain of mDC-SIGN inhibit a functional interaction with EBOV-GP. Moreover, mDC-SIGN was found be monomeric, suggesting that lack of multimerization, which is believed to be required for efficient pathogen recognition by hDC-SIGN, might be one factor that prevents binding of mDC-SIGN to EBOV-GP. Our results suggest that mDC-SIGN on murine dendritic cells is not an adequate model for pathogen interactions with hDC-SIGN.

Impact of polymorphisms in the DC-SIGNR neck domain on the interaction with pathogens.

The lectins DC-SIGN and DC-SIGNR augment infection by human immunodeficiency virus (HIV), Ebolavirus (EBOV) and other pathogens. The neck domain of these proteins drives multimerization, which is believed to be required for efficient recognition of multivalent ligands. The neck domain of DC-SIGN consists of seven sequence repeats with rare variations. In contrast, the DC-SIGNR neck domain is polymorphic and, in addition to the wild type (wt) allele with seven repeat units, allelic forms with five and six sequence repeats are frequently found. A potential association of the DC-SIGNR genotype and risk of HIV-1 infection is currently under debate. Therefore, we investigated if DC-SIGNR alleles with five and six repeat units exhibit defects in pathogen capture. Here, we show that wt DC-SIGNR and patient derived alleles with five and six repeats bind viral glycoproteins, augment viral infection and tetramerize with comparable efficiency. Moreover, coexpression of wt DC-SIGNR and alleles with five repeats did not decrease the interaction with pathogens compared to expression of each allele alone, suggesting that potential formation of hetero-oligomers does not appreciably reduce pathogen binding, at least under conditions of high expression. Thus, our results do not provide evidence for diminished pathogen capture by DC-SIGNR alleles with five and six repeat units. Albeit, we cannot exclude that subtle, but in vivo relevant differences remained undetected, our analysis suggests that indirect mechanisms could account for the association of polymorphisms in the DC-SIGNR neck region with reduced risk of HIV-1 infection.

Highly conserved regions within the spike proteins of human coronaviruses 229E and NL63 determine recognition of their respective cellular receptors.

We have recently demonstrated that the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin converting enzyme 2 (ACE2) also mediates cellular entry of the newly discovered human coronavirus (hCoV) NL63. Here, we show that expression of DC-SIGN augments NL63 spike (S)-protein-driven infection of susceptible cells, while only expression of ACE2 but not DC-SIGN is sufficient for entry into nonpermissive cells, indicating that ACE2 fulfills the criteria of a bona fide hCoV-NL63 receptor. As for SARS-CoV, murine ACE2 is used less efficiently by NL63-S for entry than human ACE2. In contrast, several amino acid exchanges in human ACE2 which diminish SARS-S-driven entry do not interfere with NL63-S-mediated infection, suggesting that SARS-S and NL63-S might engage human ACE2 differentially. Moreover, we observed that NL63-S-driven entry was less dependent on a low-pH environment and activity of endosomal proteases compared to infection mediated by SARS-S, further suggesting differences in hCoV-NL63 and SARS-CoV cellular entry. NL63-S does not exhibit significant homology to SARS-S but is highly related to the S-protein of hCoV-229E, which enters target cells by engaging CD13. Employing mutagenic analyses, we found that the N-terminal unique domain in NL63-S, which is absent in 229E-S, does not confer binding to ACE2. In contrast, the highly homologous C-terminal parts of the NL63-S1 and 229E-S1 subunits in conjunction with distinct amino acids in the central regions of these proteins confer recognition of ACE2 and CD13, respectively. Therefore, despite the high homology of these sequences, they likely form sufficiently distinct surfaces, thus determining receptor specificity.