Human Fatal Zaire Ebola Virus Infection Is Associated with an Aberrant Innate Immunity and with Massive Lymphocyte Apoptosis

Background

Ebolavirus species Zaire (ZEBOV) causes highly lethal hemorrhagic fever, resulting in the death of 90% of patients within days. Most information on immune responses to ZEBOV comes from in vitro studies and animal models. The paucity of data on human immune responses to this virus is mainly due to the fact that most outbreaks occur in remote areas. Published studies in this setting, based on small numbers of samples and limited panels of immunological markers, have given somewhat different results.

Methodology/Principal Findings

Here, we studied a unique collection of 56 blood samples from 42 nonsurvivors and 14 survivors, obtained during the five outbreaks that occurred between 1996 and 2003 in Gabon and Republic of Congo. Using Luminex technology, we assayed 50 cytokines in all 56 samples and performed phenotypic analyses by flow cytometry. We found that fatal outcome was associated with hypersecretion of numerous proinflammatory cytokines (IL-1β, IL-1RA, IL-6, IL-8, IL-15 and IL-16), chemokines and growth factors (MIP-1α, MIP-1β, MCP-1, M-CSF, MIF, IP-10, GRO-α and eotaxin). Interestingly, no increase of IFNα2 was detected in patients. Furthermore, nonsurvivors were also characterized by very low levels of circulating cytokines produced by T lymphocytes (IL-2, IL-3, IL-4, IL-5, IL-9, IL-13) and by a significant drop of CD3+CD4+ and CD3+CD8+ peripheral cells as well as a high increase in CD95 expression on T lymphocytes.

Conclusions/Significance

This work, the largest study to be conducted to date in humans, showed that fatal outcome is associated with aberrant innate immune responses and with global suppression of adaptive immunity. The innate immune reaction was characterized by a “cytokine storm,” with hypersecretion of numerous proinflammatory cytokines, chemokines and growth factors, and by the noteworthy absence of antiviral IFNα2. Immunosuppression was characterized by very low levels of circulating cytokines produced by T lymphocytes and by massive loss of peripheral CD4 and CD8 lymphocytes, probably through Fas/FasL-mediated apoptosis.     

Cysteine cathepsins S and L modulate anti-angiogenic activities of human endostatin

Human endostatin, a potent anti-angiogenic protein, is generated by release of the C terminus of collagen XVIII. Here, we propose that cysteine cathepsins are involved in both the liberation and activation of bioactive endostatin fragments, thus regulating their anti-angiogenic properties. Cathepsins B, S, and L efficiently cleaved in vitro FRET peptides that encompass the hinge region corresponding to the N terminus of endostatin. However, in human umbilical vein endothelial cell-based assays, silencing of cathepsins S and L, but not cathepsin B, impaired the generation of the ～22-kDa endostatin species. Moreover, cathepsins L and S released two peptides from endostatin with increased angiostatic properties and both encompassing the NGR sequence, a vasculature homing motif. The G10T peptide (residues 1455-1464: collagen XVIII numbering) displayed compelling anti-proliferative (EC(50) = 0.23 nm) and proapoptotic properties. G10T inhibited aminopeptidase N (APN/CD13) and reduced tube formation of endothelial cells in a manner similar to bestatin. Combination of G10T with bestatin resulted in no further increase in anti-angiogenic activity. Taken together, these data suggest that endostatin-derived peptides may represent novel molecular links between cathepsins and APN/CD13 in the regulation of angiogenesis.     

Spatio‐temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo‐vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP‐labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co‐localisation studies using GFP–CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast‐associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP–CESA from doughnut‐shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP–CESA density diminished, whereas GFP–CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP–CESA in clathrin‐containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose‐deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.     

Arabidopsis PECTIN METHYLESTERASE17 is co-expressed with and processed by SBT3.5, a subtilisin-like serine protease

Background and Aims

In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform.

Methods

Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development.

Key Results

A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm.

Conclusions

By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME–SBT pairs.     

Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal Degradation

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.     

Global imprint of historical connectivity on freshwater fish biodiversity

The relative importance of contemporary and historical processes is central for understanding biodiversity patterns. While several studies show that past conditions can partly explain the current biodiversity patterns, the role of history remains elusive. We reconstructed palaeo‐drainage basins under lower sea level conditions (Last Glacial Maximum) to test whether the historical connectivity between basins left an imprint on the global patterns of freshwater fish biodiversity. After controlling for contemporary and past environmental conditions, we found that palaeo‐connected basins displayed greater species richness but lower levels of endemism and beta diversity than did palaeo‐disconnected basins. Palaeo‐connected basins exhibited shallower distance decay of compositional similarity, suggesting that palaeo‐river connections favoured the exchange of fish species. Finally, we found that a longer period of palaeo‐connection resulted in lower levels of beta diversity. These findings reveal the first unambiguous results of the role played by history in explaining the global contemporary patterns of biodiversity.     

Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain

The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation.