Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.Ebola viruses (EBOVs) are zoonotic, enveloped negative-strand RNA viruses belonging to the family Filoviridae which cause lethal viral hemorrhagic fever in humans and nonhuman primates (47). Currently, information regarding EBOV-encoded virulence determinants remains limited. This, coupled with our lack of understanding of biochemical and structural properties of virulence factors, limits efforts to develop novel prophylactic or therapeutic approaches toward these infections.It has been proposed that EBOV-encoded mechanisms to counter innate immune responses, particularly interferon (IFN) responses, are critical to EBOV pathogenesis (7). However, a role for viral immune evasion functions in the pathogenesis of lethal EBOV infection has yet to be demonstrated. Of the eight major EBOV gene products, two viral proteins have been demonstrated to counter host IFN responses. The VP35 protein is a viral polymerase cofactor and structural protein that also inhibits IFN-α/β production by preventing the activation of interferon regulatory factor (IRF)-3 and -7 (3, 4, 8, 24, 27, 34, 41). VP35 also inhibits the activation of PKR, an IFN-induced, double-stranded RNA (dsRNA)-activated kinase with antiviral activity, and inhibits RNA silencing (17, 20, 48). The VP24 protein is a minor structural protein implicated in virus assembly and regulation of viral RNA synthesis, and changes in VP24 coding sequences are also associated with adaptation of EBOVs to mice and guinea pigs (2, 13, 14, 27, 32, 37, 50, 52). Further, VP24 inhibits cellular responses to both IFN-α/β and IFN-γ by preventing the nuclear accumulation of tyrosine-phosphorylated STAT1 (44, 45). The functions of VP35 and VP24 proteins are manifested in EBOV-infected cells by the absence of IRF-3 activation, impaired production of IFN-α/β, and severely reduced expression of IFN-induced genes, even after treatment of infected cells with IFN-α (3, 19, 21, 22, 24, 25, 28).Previous studies proposed that VP35 basic residues 305, 309, and 312 are required for VP35 dsRNA binding activity (26). VP35 residues K309 and R312 were subsequently identified as critical for binding to dsRNA, and mutation of these residues impaired VP35 suppression of IFN-α/β production (8). In vivo, an EBOV engineered to carry a VP35 R312A point mutation exhibited reduced replication in mice (23). However, because the parental recombinant EBOV into which the mutation was built did not cause disease in these animals, the impact of the mutation on viral pathogenesis could not be fully evaluated. Further, the lack of available structural and biochemical data to explain how the R312A mutation affects VP35 function limited avenues for the therapeutic targeting of critical VP35 functions. Recent structural analyses of the VP35 carboxy-terminal interferon inhibitory domain (IID) suggested that additional residues from the central basic patch may contribute to VP35 dsRNA binding activity and IFN-antagonist function (30). However, a direct correlation between dsRNA and IFN inhibitory functions of VP35 with viral pathogenesis is currently lacking.In order to further define the molecular basis for VP35 dsRNA binding and IFN-antagonist function and to define the contribution of these functions to EBOV pathogenesis, an integrated molecular, structural, and virological approach was taken. The data presented below identify two VP35 carboxy-terminal basic amino acids, K319 and R322, as required for its dsRNA binding and IFN-antagonist functions. Interestingly, these residues are outside the region originally identified as being important for dsRNA binding and IFN inhibition (26). However, they lie within the central basic patch identified by prior structural studies (26, 30). Introduction of these mutations (VP35 with these mutations is designated KRA) into recombinant EBOV renders this otherwise fully lethal virus avirulent in guinea pigs. KRA-infected animals also develop EBOV-specific antibodies and become fully resistant to subsequent challenge with wild-type (WT) virus. Our data further reveal that the KRA EBOV is immunogenic and likely replicates to low levels early after infection in vivo. However, the mutant virus is subsequently cleared by host immune responses. These data demonstrate that the VP35 central basic patch is important not only for IFN-antagonist function but also for EBOV immune evasion and pathogenesis in vivo. High-resolution structural analysis, coupled with our in vitro and in vivo analyses of the recombinant Ebola viruses, provides the molecular basis for loss of function by the VP35 mutant and highlights the therapeutic potential of targeting the central basic patch with small-molecule inhibitors and for future vaccine development efforts.     

Development of functional B cells in a line of SCID mice with transgenes coding for anti-double-stranded DNA antibody

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vkappa8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVkappa8 SCID mice) generally lack serum Ig. However, 56RVkappa8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH-/- donors. Thus, anti-dsDNA B cells that escape deletion in 56RVkappa8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.     

Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.     

Comparison of expression systems for the production of human interferon-α2b

The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 μg/L of culture, while the amount of nuclear expression in the plant was 4.46 μg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.     

Prevalence and phylogenetic relationship of two β-carbonic anhydrases in affiliates of Enterobacteriaceae

Enterobacteriaceae, one of the major families of microorganisms that inhabit the soil and gut, internally regulate constant fluctuations in soil and gut pH by buffering these changes through the presence of carbonic anhydrase (CA). In our study, we prove the prevalence of β-CA, derived from the can gene, in members of Enterobacteriaceae by using a combination of experimental and bioinformatics approaches. Enzyme purification and western blot analysis revealed the presence of β-CA in Enterobacter sp. RS1. Genetic studies confirmed the presence of β-CA in both Enterobacter sp. RS1 and Citrobacter freundii SW3. Our analysis of the divergence of cynT and can genes among harboring members indicated that the can gene was more prominent in Enterobacteriaceae than cynT. Sequence analysis of the can gene revealed a >25 % similarity among all sequences and a >50 % similarity among sequences from the Enterobacteriaceae family. The β-CA from C. freundii SW3 and Enterobacter sp. RS1, isolated from soil and used in this study, possessed a high similarity with the can gene. The close association among Enterobacteriaceae genera usually found in the soil and gut and the sequence similarity of β-CA in the different genera of Enterobacteriaceae suggest the importance of the can gene in oscillating environmental conditions.     

A sticky situation: the unexpected stability of malaria elimination

Malaria eradication involves eliminating malaria from every country where transmission occurs. Current theory suggests that the post-elimination challenges of remaining malaria-free by stopping transmission from imported malaria will have onerous operational and financial requirements. Although resurgent malaria has occurred in a majority of countries that tried but failed to eliminate malaria, a review of resurgence in countries that successfully eliminated finds only four such failures out of 50 successful programmes. Data documenting malaria importation and onwards transmission in these countries suggests malaria transmission potential has declined by more than 50-fold (i.e. more than 98%) since before elimination. These outcomes suggest that elimination is a surprisingly stable state. Elimination''s ‘stickiness’ must be explained either by eliminating countries starting off qualitatively different from non-eliminating countries or becoming different once elimination was achieved. Countries that successfully eliminated were wealthier and had lower baseline endemicity than those that were unsuccessful, but our analysis shows that those same variables were at best incomplete predictors of the patterns of resurgence. Stability is reinforced by the loss of immunity to disease and by the health system''s increasing capacity to control malaria transmission after elimination through routine treatment of cases with antimalarial drugs supplemented by malaria outbreak control. Human travel patterns reinforce these patterns; as malaria recedes, fewer people carry malaria from remote endemic areas to remote areas where transmission potential remains high. Establishment of an international resource with backup capacity to control large outbreaks can make elimination stickier, increase the incentives for countries to eliminate, and ensure steady progress towards global eradication. Although available evidence supports malaria elimination''s stickiness at moderate-to-low transmission in areas with well-developed health systems, it is not yet clear if such patterns will hold in all areas. The sticky endpoint changes the projected costs of maintaining elimination and makes it substantially more attractive for countries acting alone, and it makes spatially progressive elimination a sensible strategy for a malaria eradication endgame.     

Heat tolerance in rice mutants is associated with reduced accumulation of reactive oxygen species

Four mutants induced by ethylmethane sulphonate (N22-H-dgl56, N22-H-dgl101, N22-H-dgl162 and N22-H-dgl219) with conspicuous dark green leaves were identified in the drought and heat-tolerant rice cultivar Nagina22 (N22), when screened under prolonged drought and heat conditions in field. During dark-induced senescence, these mutants maintained higher chlorophyll and carotenoid contents, and photochemical efficiency of photosystem 2 in comparison with N22. Following heat treatment, these mutants accumulated less reactive oxygen species (assayed by histochemical staining for H₂O₂ and superoxide radicals) and maintained higher chlorophyll content than N22.     

Release mode of large and small dense-core vesicles specified by different synaptotagmin isoforms in PC12 cells

Many cells release multiple substances in different proportions according to the specific character of a stimulus. PC12 cells, a model neuroendocrine cell line, express multiple isoforms of the exocytotic Ca(2+) sensor synaptotagmin. We show that these isoforms sort to populations of dense-core vesicles that differ in size. These synaptotagmins differ in their Ca(2+) sensitivities, their preference for full fusion or kiss-and-run, and their sensitivity to inhibition by synaptotagmin IV. In PC12 cells, vesicles that harbor these different synaptotagmin isoforms can be preferentially triggered to fuse by different forms of stimulation. The mode of fusion is specified by the synaptotagmin isoform activated, and because kiss-and-run exocytosis can filter small molecules through a size-limiting fusion pore, the activation of isoforms that favor kiss-and-run will select smaller molecules over larger molecules packaged in the same vesicle. Thus synaptotagmin isoforms can provide multiple levels of control in the release of different molecules from the same cell.     

Improved mannan-degrading enzymes' production by Aspergillus niger through medium optimization

The effect of different carbon and nitrogen sources on the production of mannan-degrading enzymes, focussing on β-mannanase, by Aspergillus niger was investigated using shake flask culture. The β-mannanase activity obtained during growth of A. niger on guar gum (GG, 1495 nkat mL(-1)) was much higher than those observed on other carbon substrates, locust bean gum (1148 nkat mL(-1)), α-cellulose (10.7 nkat mL(-1)), glucose (8.8 nkat mL(-1)) and carboxymethylcellulose (4.6 nkat mL(-1)). For fermentation using GG as a carbon source, bacteriological peptone gave the highest β-mannanase activity (1744 nkat mL(-1)) followed by peptone from meat (1168 nkat mL(-1)), yeast extract (817 nkat mL(-1)), ammonium sulphate (241 nkat mL(-1)), ammonium nitrate (113 nkat mL(-1)) and ammonium chloride (99 nkat mL(-1)) when used as a nitrogen source. The composition of bacteriological peptone and initial pH of the medium were further optimized using response surface methodology (RSM). Medium consisted of 21.3 g L(-1) GG and 57 g L(-1) peptone with initial culture pH of 5.5 was optimum for β-mannanase production (2063 nkat mL(-1)) by A. niger. The β-mannanase production obtained in this study using A. niger was significantly higher than those reported in the literature.