A mutation in the Ebola virus envelope glycoprotein restricts viral entry in a host species- and cell-type-specific manner.

Zaire Ebola virus (EBOV) is a zoonotic pathogen that causes severe hemorrhagic fever in humans. A single viral glycoprotein (GP) mediates viral attachment and entry. Here, virus-like particle (VLP)-based entry assays demonstrate that a GP mutant, GP-F88A, which is defective for entry into a variety of human cell types, including antigen-presenting cells (APCs), such as macrophages and dendritic cells, can mediate viral entry into mouse CD11b(+) APCs. Like that of wild-type GP (GP-wt), GP-F88A-mediated entry occurs via a macropinocytosis-related pathway and requires endosomal cysteine proteases and an intact fusion peptide. Several additional hydrophobic residues lie in close proximity to GP-F88, including L111, I113, L122, and F225. GP mutants in which these residues are mutated to alanine displayed preferential and often impaired entry into several cell types, although not in a species-specific manner. Niemann-Pick C1 (NPC1) protein is an essential filovirus receptor that binds directly to GP. Overexpression of NPC1 was recently demonstrated to rescue GP-F88A-mediated entry. A quantitative enzyme-linked immunosorbent assay (ELISA) demonstrated that while the F88A mutation impairs GP binding to human NPC1 by 10-fold, it has little impact on GP binding to mouse NPC1. Interestingly, not all mouse macrophage cell lines permit GP-F88A entry. The IC-21 cell line was permissive, whereas RAW 264.7 cells were not. Quantitative reverse transcription (RT)-PCR assays demonstrate higher NPC1 levels in GP-F88A permissive IC-21 cells and mouse peritoneal macrophages than in RAW 264.7 cells. Cumulatively, these studies suggest an important role for NPC1 in the differential entry of GP-F88A into mouse versus human APCs.

Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles.

Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean-Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

The exosporium of B. cereus contains a binding site for gC1qR/p33: implication in spore attachment and/or entry.

B. cereus, is a member of a genus of aerobic, gram-positive, spore-forming rod-like bacilli, which includes the deadly, B. anthracis. Preliminary experiments have shown that gC1qR binds to B. cereus spores that have been attached to microtiter plates. The present studies were therefore undertaken, to examine if cell surface gC1qR plays a role in B. cereus spore attachment and/or entry. Monolayers of human colon carcinoma (Caco-2) and lung cells were grown to confluency on 6 mm coverslips in shell vials with gentle swirling in a shaker incubator. Then, 2 microl of a suspension of strain SB460 B. cereus spores (3x10(8)/ml, in sterile water), were added and incubated (1-4 h; 36 degrees C) in the presence or absence of anti-gC1qR mAb-carbon nanoloops. Examination of these cells by EM revealed that: (1) When B. cereus endospores contacted the apical Caco-2 cell surface, or lung cells, gC1qR was simultaneously detectable, indicating upregulation of the molecule. (2) In areas showing spore contact with the cell surface, gC1qR expression was often adjacent to the spores in association with microvilli (Caco-2 cells) or cytoskeletal projections (lung cells). (3) Furthermore, the exosporia of the activated and germinating spores were often decorated with mAb-nanoloops. These observations were further corroborated by experiments in which B.cereus spores were readily taken up by monocytes and neutrophils, and this uptake was partially inhibited by mAb 60.11, which recognizes the C1q binding site on gC1qR. Taken together, the data suggest a role, for gC1qR at least in the initial stages of spore attachment and/or entry.

gC1qR/p33 serves as a molecular bridge between the complement and contact activation systems and is an important catalyst in inflammation.

The receptor for the globular heads of C1q, gC1qR/p33, is a ubiquitously expressed protein, which is distributed both intracellularly and on the cell-surface protein. In addition to C1q, this molecule also is able to bind several other biologically important plasma ligands, including high-molecular-weight kininogen (HK), factor XII (FXII), and multimeric vitronectin. Previous studies have shown that incubation of FXII, prekallikrein, and HK with gC1qR leads to a zinc-dependent and FXII-dependent conversion of prekallikrein to kallikrein, a requisite for kinin generation. In addition, these studies showed that normal plasma, but not plasma deficient in FXII, PK, or HK, activate upon binding to endothelial cells (EC), and that this activation could be inhibited by antibody to gClqR. In these studies, we show that incubation of serum with microtiter plate bound gC1qR results in complement activation, as evidenced by the binding and activation of C1 and generation of C4d. However, neither Clq-deficient serum nor a truncated form of gC1qR (gC1qRA74-96), supported complement activation. Taken together, the data strongly suggest that at sites of inflammation, such as vasculitis and atherosclerosis, where gC1qR as well as its two important plasma ligands, C1q and HK, have been shown to be simultaneously present, soluble or cell-surface-expressed gC1qR may contribute to the inflammatory process by modulating complement activation, kinin generation, and perhaps even initiation of clotting via the contact system. Based on these and other published data, we propose a model of inflammation in which atherogenic factors (e.g., immune complexes, virus, or bacteria) are perceived not only to convert the endothelium into a procoagulant and proinflammatory surface, but also to induce enhanced expression of cell surface molecules such as gC1qR. Enhanced expression of gC1qR in turn leads to: (i) high-affinity C1q binding and cell production of proinflammatory factors, and (ii) high-affinity HK binding and facilitation of the assembly of contact activation proteins leading to generation of bradykinin and possibly coagulation through activation of FXI.

Intracellular calcium release is required for caspase-3 and -9 activation.

Increase in intracellular Ca2+ [Ca2+]i regulates many biological functions including apoptosis, but the protein(s) linking [Ca2+]i and apoptosis are not completely understood. We have previously shown that IP3R-deficient cells are resistant to T-cell receptor (TCR)-induced apoptosis due to lack of Ca2+ release from endoplasmic reticulum (ER) and calcineurin activation. Here we show that caspase-9 and -3 are not activated in IP3R-deficient cells after TCR stimulation, consistent with the resistance of these cells to apoptosis. However, we also demonstrate that Bcl-2 expression in IP3R-deficient cells is comparable to control cells. Taken together, these results strongly suggest that IP3R-mediated Ca2+ release plays a critical role in regulating the activity of caspases-3 and -9 independent of Bcl-2.