A Forward Genetic Strategy Reveals Destabilizing Mutations in the Ebolavirus Glycoprotein That Alter Its Protease Dependence during Cell Entry

Ebolavirus (EBOV) entry into cells requires proteolytic disassembly of the viral glycoprotein, GP. This proteolytic processing, unusually extensive for an enveloped virus entry protein, is mediated by cysteine cathepsins, a family of endosomal/lysosomal proteases. Previous work has shown that cleavage of GP by cathepsin B (CatB) is specifically required to generate a critical entry intermediate. The functions of this intermediate are not well understood. We used a forward genetic strategy to investigate this CatB-dependent step. Specifically, we generated a replication-competent recombinant vesicular stomatitis virus bearing EBOV GP as its sole entry glycoprotein and used it to select viral mutants resistant to a CatB inhibitor. We obtained mutations at six amino acid positions in GP that independently confer complete resistance. All of the mutations reside at or near the GP1-GP2 intersubunit interface in the membrane-proximal base of the prefusion GP trimer. This region forms a part of the “clamp” that holds the fusion subunit GP2 in its metastable prefusion conformation. Biochemical studies suggest that most of the mutations confer CatB independence not by altering specific cleavage sites in GP but rather by inducing conformational rearrangements in the prefusion GP trimer that dramatically enhance its susceptibility to proteolysis. The remaining mutants did not show the preceding behavior, indicating the existence of multiple mechanisms for acquiring CatB independence during entry. Altogether, our findings suggest that CatB cleavage is required to facilitate the triggering of viral membrane fusion by destabilizing the prefusion conformation of EBOV GP.Filoviruses are enveloped, filamentous, nonsegmented negative-sense RNA viruses that can cause a deadly hemorrhagic fever with case fatality rates in excess of 90% (see references 4, 20, and 37 for recent reviews). All known filoviruses belong to one of two genera: Ebolavirus (EBOV), consisting of the five species Zaire (ZEBOV), Côte d''Ivoire, Sudan, Reston, and Bundibugyo (tentative); and Marburgvirus, consisting of the single Lake Victoria species (21, 62).Cell entry by filoviruses is mediated by their envelope glycoprotein, GP (60, 68). Mature GP is a trimer of three disulfide-linked GP1-GP2 heterodimers. GP1 and GP2 are generated by endoproteolytic cleavage of the GP0 precursor polypeptide by a furin-like protease during transport to the cell surface (31, 39, 63, 69). The membrane-distal subunit, GP1, mediates viral adhesion to host cells (10, 18, 38, 42, 56, 59) and regulates the activity of the transmembrane subunit, GP2, which catalyzes fusion of viral and cellular membrane bilayers (30, 39, 41, 64, 65). The consequence of membrane fusion is cytoplasmic delivery of the viral nucleocapsid cargo.Lee et al. (39) recently solved the crystal structure of a ZEBOV GP prefusion trimer lacking the heavily glycosylated GP1 mucin domain (Muc) and the GP2 transmembrane domain (see Fig. ​Fig.5).5). The three GP1 subunits together form a bowl-like structure encircled by sequences from the three GP2 subunits. The trimer is held together by GP1-GP2 and GP2-GP2 contacts; the hydrophobic GP2 fusion loop packs against the external surface of adjacent GP1 subunits, and each GP2 subunit contributes a strand to a trimeric α-helical coiled-coil stem. GP1 is organized into three subdomains. The base is intimately associated with GP2 and clamps it in its prefusion conformation. The head is proposed to mediate virus receptor binding during entry (10, 18, 38, 42). The glycan cap resides at the top of the trimer and is critical for GP assembly but must be removed during entry (see below) (31, 42). The base and glycan cap are connected by the β13-β14 loop, which was not visualized in the structure. The location and structure of the Muc domain are also unknown, but it is proposed to sheathe the top and/or sides of the prefusion GP trimer (39). Muc is dispensable for ZEBOV GP-dependent entry in tissue culture but may play roles in virus-cell adhesion and immune evasion in vivo (31, 42, 44, 56, 59).Open in a separate windowFIG. 5.CA074^R mutations localize at or near the GP1-GP2 interface in the GP prefusion crystal structure. In all diagrams, GP1 is depicted in blue, GP2 in red, GP1 CA074^R mutations in green, and GP2 CA074^R mutations in yellow. (A) Linear representation of the amino acid sequence of GPΔMuc. S-S indicates the intersubunit disulfide bond between C53 and C609. sp, signal peptide; fl, fusion loop; hr1 and hr2, heptad repeats; tm, transmembrane domain; N, N terminus; C, C terminus. (B) Structure of GP in a prefusion conformation (39). Cartoon representation of a GP1-GP2 monomer is shown. Remaining subunits are shown as a surface-shaded watermark. The boxed inset contains the membrane-proximal base of the trimer, in which the CA074^R mutations are located. The β13-β14 loop is modeled as a chain of blue circles. (C) Magnified view of the inset shown in panel B rotated by 90°. The side chains of D47, I584, K588, and their contacting residues are shown. Dashed pink lines connect atoms from different side chains separated by ≤3.9 Å. Other CA074^R residues are not shown for clarity. (D) View shown in panel C rotated by 90°. (E) Schematic diagram of the potential interactions made by residues mutated in the CA074^R viruses. Residues approaching ≤3.9 Å to each CA074^R residue are shown. Beige arcs, hydrophobic interactions; dashed lines, potential ionic interactions. Visualizations of the GP structures shown in panels B to D (Protein Data Bank accession no. 3CSY) were rendered in Pymol (Delano Scientific).Crystal structures of ZEBOV GP2 in its postfusion conformation indicate that filovirus GP is a “class I” viral membrane fusion protein (41, 65). Like the prototypic class I fusion proteins of human immunodeficiency virus and influenza virus, GP2 contains a hydrophobic fusion peptide near its N terminus and N- and C-terminal α-helical heptad repeat sequences (HR1 and HR2, respectively) (22, 28, 30, 39, 41, 64, 65, 67) (see Fig. ​Fig.5).5). GP2 drives membrane fusion by undergoing large-scale conformational changes; the prefusion HR1 helix-loop-helix rearranges to an unbroken α-helix, projecting the fusion loop into the endosomal membrane, and GP2 jackknifes on itself to form a hairpin-like structure in which the HR2s pack against grooves in the trimeric HR1 coiled coil (41, 65).The available GP structures make clear that the transition of GP2 from prefusion to postfusion conformation requires its release from its binding groove in the GP1 base subdomain. For all known class I fusion proteins, this transition is controlled by priming and triggering events. Priming typically involves a single endoproteolytic cleavage of the glycoprotein mediated by a cellular protease within the secretory pathway of the virus-producer cells (e.g., human immunodeficiency virus ENV → SU + TM by furin [27]). This cleavage is essential because it liberates an N-terminal fusion peptide and allows the glycoprotein to rearrange during fusion. Unusually for a class I fusion glycoprotein, however, ZEBOV GP does not require cleavage to GP1 and GP2 by a furin-like protease, even though this cleavage occurs efficiently (46, 69). Instead, the GP trimer is primed by extensive proteolytic remodeling during entry. This process is mediated by cysteine cathepsins, a class of papain superfamily cysteine proteases active within the cellular endosomal/lysosomal pathway (14, 54).The cysteine cathepsins B (CatB) and L (CatL) play essential and accessory roles, respectively, in ZEBOV entry into Vero cells (14). The functions of these enzymes in viral entry can be recapitulated in vitro. Incubation of vesicular stomatitis virus (VSV) pseudotypes bearing ZEBOV GP (VSV-GP) with a mixture of purified human CatL and CatB, or with the bacterial protease thermolysin (THL), results in the cleavage and removal of GP1 Muc and glycan cap sequences, leaving a stable ∼17-kDa N-terminal GP1 fragment and intact GP2 (see Fig. ​Fig.5)5) (18, 54). VSV particles containing this GP_17K intermediate no longer require CatB activity within cells, strongly suggesting that this protease plays a critical role in generating a related primed species during viral entry (54). Strikingly, incubation of VSV-GP with CatL alone (14, 54) or with bovine chymotrypsin (CHT) (this study) (Fig. ​(Fig.1;1; see also Fig. ​Fig.7)7) generates a similar but distinct GP_18K intermediate (containing a slightly larger ∼18-kDa GP1 fragment) that cannot bypass the requirement for CatB during entry. Therefore, the removal of a few residues from GP_18K by CatB is crucial for viral entry. The reason for this requirement is unknown. Finally, VSV-GP_17K particles cannot infect cells completely devoid of cysteine cathepsin activity, indicating the existence of at least one additional cysteine protease-dependent step during entry (34, 54; present study). The signal that acts on a fully primed GP intermediate to trigger membrane fusion remains unknown.Open in a separate windowFIG. 1.CatB activity is required for entry of ZEBOV GP-dependent entry, whereas CatL activity is dispensable. Vero cells were pretreated for 4 h with 1% (vol/vol) DMSO (vehicle), 0.5 μM FYdmk (CatL-selective inhibitor), 80 μM CA074 (CatB-selective inhibitor), 0.5 μM FYdmk plus 80 μM CA074, or 300 μM E64 (pan-cysteine cathepsin inhibitor). (A) The cells were then challenged with VSV-GPΔMuc, CHT-derived VSV-GP_18K (CHT-GP_18K), THL-derived VSV-GP_17K (THL-GP_17K), or VSV-G pseudotypes at a low MOI (0.02 to 0.1 eGFP-positive infectious units [iu] per cell) in the presence of drug, and viral titers (iu/ml) were determined at 18 h postinfection. CatB and CatL activities in extracts prepared from a parallel set of pretreated cells were measured by fluorogenic peptide turnover and are shown (bottom). Averages ± standard deviations (SD) for six trials from three independent experiments are shown. CatL activity below the detection threshold is indicated as zero without an accompanying SD. (B) Vero cells pretreated with protease inhibitors were challenged with VSV-GPΔMuc, cathepsin L-derived VSV-GP_18K (CatL-GP_18K), or cathepsin B-derived VSV-GP_17K (CatB-GP_17K), and viral infectivity was measured as described above. Averages ± SD for three trials from a representative experiment are shown.Open in a separate windowFIG. 7.rVSV-GPΔMuc mutants resemble the WT in cleavage to GP_18K and GP_17K intermediates. WT or mutant rVSV-GPΔMuc was incubated with the indicated protease(s) as described in Materials and Methods and then deglycosylated with PNGaseF (except for N40K and T42A, which lack the N40 glycan and do not require deglycosylation at this position). The resulting GP1 proteolytic fragments were resolved by SDS-PAGE and detected by Western blotting. Shorter protease incubation times were necessary to obtain cleavage intermediates for some mutants (see text for details). Positions of uncleaved GP1 and the ∼18-kDa and ∼17-kDa cleavage fragments are indicated on the left. *, partially cleaved GP fragment of unknown composition. Experimental samples shown on each gel (bold labels) were flanked by WT samples cleaved with CHT (WT 18K) or THL (WT 17K) to provide markers of band mobility.In this study, we used a forward genetic strategy to investigate the CatB-dependent step in ZEBOV entry. Specifically, we engineered and rescued a recombinant VSV (rVSV) encoding a mucin domain-deleted ZEBOV GP in place of the VSV glycoprotein G and used it to select viral mutants resistant to the CatB inhibitor CA074. Analysis of these viruses identified mutations in both GP1 and GP2 that allow CatB-independent cell entry. We found that GP_18K and/or GP_17K intermediates derived from some but not all of the mutant GPs are conformationally distinct from the wild type (WT), suggesting the existence of multiple mechanisms for CA074 resistance. Taken together, our results indicate that ZEBOV GP→GP_17K cleavage by CatB promotes fusion triggering and viral entry by destabilizing the prefusion conformation of GP.     

Determining selectivity of phosphoinositide-binding domains

The burgeoning of phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily than others. With a focus on the methods used in our laboratory, we describe here the considerations that need to be taken into account when establishing-and quantitating-the specific binding of a protein or domain to phosphoinositides in membranes. We also discuss briefly a few examples in which no clear consensus has yet been reached as to the specificity of a given domain or protein because of discrepancies between different commonly used approaches.     

Juxtaposed half-cystines as disulphide bridged partners in protein tertiary structure

Disulphide bridges involving juxtaposed half-cystines are observed in a number of protein three-dimensional structures analyzed from the Protein Data Bank. These disulphide bridges comprise a 'ring of 8-atoms' corresponding to Calpha1-C'-N-Calpha2-Cbeta2-Sgamma2-Sgamma1-Cbeta1-Calpha1 in the two half-cystines. The presence of such disulphide bridges introduces a 'bend' or 'kink' in the protein polypeptide chain.     

Association between Farming and Chronic Energy Deficiency in Rural South India

Objective

To examine factors associated with chronic energy deficiency (CED) and anaemia in disadvantaged Indian adults who are mostly involved in subsistence farming.

Design

A cross-sectional study in which we collected information on socio-demographic factors, physical activity, anthropometry, blood haemoglobin concentration, and daily household food intake. These data were used to calculate body mass index (BMI), basal metabolic rate (BMR), daily energy expenditure, and energy and nutrient intake. Multivariable backward stepwise logistic regression was used to assess socioeconomic and lifestyle factors associated with CED (defined as BMI<18 kg/m²) and anaemia.

Setting

The study was conducted in 12 villages, in the Rishi Valley, Andhra Pradesh, India.

Subjects

Individuals aged 18 years and above, residing in the 12 villages, were eligible to participate.

Results

Data were available for 1178 individuals (45% male, median age 36 years (inter quartile range (IQR 27–50)). The prevalence of CED (38%) and anaemia (25%) was high. Farming was associated with CED in women (2.20, 95% CI: 1.39–3.49) and men (1.71, 95% CI: (1.06–2.74). Low income was also significantly associated with CED, while not completing high school was positively associated with anaemia. Median iron intake was high: 35.7 mg/day (IQR 26–46) in women and 43.4 mg/day (IQR 34–55) in men.

Conclusions

Farming is an important risk factor associated with CED in this rural Indian population and low dietary iron is not the main cause of anaemia. Better farming practice may help to reduce CED in this population.     

Bioremediation of hexavalent chromium from wastewater using bacteria-a green technology

Biodegradation - Hexavalent chromium has high toxic effect on the ecological system. The aim of the present study is to isolate and characterize the bacteria that can reduce the toxicity of...     

Species importance in a heterospecific foraging association network

There is a growing recognition of the need to integrate non‐trophic interactions into ecological networks for a better understanding of whole‐community organization. To achieve this, the first step is to build networks of individual non‐trophic interactions. In this study, we analyzed a network of interdependencies among bird species that participated in heterospecific foraging associations (flocks) in an evergreen forest site in the Western Ghats, India. We found the flock network to contain a small core of highly important species that other species are strongly dependent on, a pattern seen in many other biological networks. Further, we found that structural importance of species in the network was strongly correlated to functional importance of species at the individual flock level. Finally, comparisons with flock networks from other Asian forests showed that the same taxonomic groups were important in general, suggesting that species importance was an intrinsic trait and not dependent on local ecological conditions. Hence, given a list of species in an area, it may be possible to predict which ones are likely to be important. Our study provides a framework for the investigation of other heterospecific foraging associations and associations among species in other non‐trophic contexts.     

C-peptide inhibitors of Ebola virus glycoprotein-mediated cell entry: Effects of conjugation to cholesterol and side chain–side chain crosslinking

We previously described potent inhibition of Ebola virus entry by a ‘C-peptide’ based on the GP2 C-heptad repeat region (CHR) targeted to endosomes (‘Tat-Ebo’). Here, we report the synthesis and evaluation of C-peptides conjugated to cholesterol, and Tat-Ebo analogs containing covalent side chain–side chain crosslinks to promote α-helical conformation. We found that the cholesterol-conjugated C-peptides were potent inhibitors of Ebola virus glycoprotein (GP)-mediated cell entry (～10³-fold reduction in infection at 40 μM). However, this mechanism of inhibition is somewhat non-specific because the cholesterol-conjugated peptides also inhibited cell entry mediated by vesicular stomatitis virus glycoprotein G. One side chain–side chain crosslinked peptide had moderately higher activity than the parent compound Tat-Ebo. Circular dichroism revealed that the cholesterol-conjugated peptides unexpectedly formed a strong α-helical conformation that was independent of concentration. Side chain–side chain crosslinking enhanced α-helical stability of the Tat-Ebo variants, but only at neutral pH. These result provide insight into mechanisms of C-peptide inhibiton of Ebola virus GP-mediated cell entry.