Novel antiviral strategies to combat human Arenavirus infections

Arenaviruses merit significant attention both as tractable model systems to study acute and persistent viral infections, and as clinically important human pathogens. Evidence indicates that LCMV remains present in the USA and Europe and capable of causing significant morbidity in infected individuals, likely being a neglected human pathogen. Moreover, new arenaviruses are being discovered in the Americas on the average of one every three years, with some of them causing severe hemorrhagic fever. In addition, weaponized forms of these viruses pose a real threat as agents of bioterrorism. Therefore, it is important to develop effective vaccines and better antiviral drugs to combat the dual threats of naturally occurring and intentionally introduced Arenavirus infections. The development of arenavirus reverse genetic systems is allowing investigators to conduct a detailed molecular characterization of the viral cis-acting signals and trans-acting factors that control each of the steps of the Arenavirus life cycle, including RNA synthesis, packaging and budding. We will discuss how this new knowledge is facilitating the establishment of novel assays to identify and characterize compounds capable of interfering with specific steps of the virus life cycle. Likewise, the ability to generate predetermined specific mutations within the arenavirus genome, and analyze their phenotypic expression, would significantly contribute to the elucidation of arenavirus-host interactions, including the bases of their ability to persist, as well as to cause severe HF (hemorrhagic fever) disease in humans. These approaches could also lead to the development of novel potent and safe Arenavirus vaccines.     

Bacterial cyanide oxygenase is a suite of enzymes catalyzing the scavenging and adventitious utilization of cyanide as a nitrogenous growth substrate

Cyanide oxygenase (CNO) from Pseudomonas fluorescens NCIMB 11764 catalyzes the pterin-dependent oxygenolytic cleavage of cyanide (CN) to formic acid and ammonia. CNO was resolved into four protein components (P1 to P4), each of which along with a source of pterin cofactor was obligately required for CNO activity. Component P1 was characterized as a multimeric 230-kDa flavoprotein exhibiting the properties of a peroxide-forming NADH oxidase (oxidoreductase) (Nox). P2 consisted of a 49.7-kDa homodimer that showed 100% amino acid identity at its N terminus to NADH peroxidase (Npx) from Enterococcus faecalis. Enzyme assays further confirmed the identities of both Nox and Npx enzymes (specific activity, 1 U/mg). P3 was characterized as a large oligomeric protein (approximately 300 kDa) that exhibited cyanide dihydratase (CynD) activity (specific activity, 100 U/mg). Two polypeptides of 38 kDa and 43 kDa were each detected in the isolated enzyme, the former believed to confer catalytic activity based on its similar size to other CynD enzymes. The amino acid sequence of an internal peptide of the 43-kDa protein was 100% identical to bacterial elongation factor Tu, suggesting a role as a possible chaperone in the assembly of CynD or a multienzyme CNO complex. The remaining P4 component consisted of a 28.9-kDa homodimer and was identified as carbonic anhydrase (specific activity, 2,000 U/mg). While the function of participating pterin and the roles of Nox, Npx, CynD, and CA in the CNO-catalyzed scavenging of CN remain to be determined, this is the first report describing the collective involvement of these four enzymes in the metabolic detoxification and utilization of CN as a bacterial nitrogenous growth substrate.     

Subplasmalemmal mitochondria modulate the activity of plasma membrane Ca2+-ATPases

Mitochondria are dynamic organelles that modulate cellular Ca2+ signals by interacting with Ca2+ transporters on the plasma membrane or the endoplasmic reticulum (ER). To study how mitochondria dynamics affects cell Ca2+ homeostasis, we overexpressed two mitochondrial fission proteins, hFis1 and Drp1, and measured Ca2+ changes within the cytosol and the ER in HeLa cells. Both proteins fragmented mitochondria, decreased their total volume by 25-40%, and reduced the fraction of subplasmalemmal mitochondria by 4-fold. The cytosolic Ca2+ signals elicited by histamine were unaltered in cells lacking subplasmalemmal mitochondria as long as Ca2+ was present in the medium, but the signals were significantly blunted when Ca2+ was removed. Upon Ca2+ withdrawal, the free ER Ca2+ concentration decreased rapidly, and hFis1 cells were unable to respond to repetitive histamine stimulations. The loss of stored Ca2+ was due to an increased activity of plasma membrane Ca2+-ATPase (PMCA) pumps and was associated with an increased influx of Ca2+ and Mn2+ across store-operated Ca2+ channels. The increased Ca2+ influx compensated for the loss of stored Ca2+, and brief Ca2+ additions between successive agonist stimulations fully corrected subsequent histamine responses. We propose that the lack of subplasmalemmal mitochondria disrupts the transfer of Ca2+ from plasma membrane channels to the ER and that the resulting increase in subplasmalemmal [Ca2+] up-regulates the activity of PMCA. The increased Ca2+ extrusion promotes ER depletion and the subsequent activation of store-operated Ca2+ channels. Cells thus adapt to the lack of subplasmalemmal mitochondria by relying on external rather than on internal Ca2+ for signaling.     

Structural insights into the secretin PulD and its trypsin-resistant core

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.     

Common features in the functional surface of scorpion beta-toxins and elements that confer specificity for insect and mammalian voltage-gated sodium channels

Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.     

Structural and biochemical characterization of a quinol binding site of Escherichia coli nitrate reductase A

The crystal structure of Escherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol has been determined to 2.0 A of resolution. We have shown that pentachlorophenol is a potent inhibitor of quinol:nitrate oxidoreductase activity and that it also perturbs the EPR spectrum of one of the hemes located in the membrane anchoring subunit (NarI). This new structural information together with site-directed mutagenesis data, biochemical analyses, and molecular modeling provide the first molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI. A possible proton conduction pathway linked to electron transfer reactions has also been defined, providing fundamental atomic details of ubiquinol oxidation by NarGHI at the bacterial membrane.     

Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.     

An alternative high yielding and highly stereoselective method for preparing an alpha-Neu5NAc-(2,6)-D-GalN3 building block suitable for further glycosylation

This paper deals with new approaches to alpha-Neu5NAc-(2,6)-D-GalN3 building blocks, suitable as glycosylation donors. The major improvement, by comparison with the results of the literature, lies in the glycosylation step of a new d-galactosamine acceptor (tert-butyldimethylsilyl 3-O-acetyl-2-azido-2-deoxy-beta-D-galactopyranoside) with O-methyl-S-[methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto-non-2-ulopyranosyl)onate] dithiocarbonate as the N-acetylneuraminic acid donor. The reaction affords the expected disaccharide in high yield (85%) and a complete alpha-Neu5NAc stereoselectivity. A subsequent oxidation step, eliminating the glycal by-product allows an easier purification. Afterwards, the tert-butyldimethylsilyl disaccharide can be transformed into a donor, after cleavage of the anomeric group in smooth conditions.     

Impact of melon accessions resistant to aphids on the demographic potential of silverleaf whitefly

Bemisia tabaci (Gennadius) and Aphis gossypii Glover are devastating melon, Cucumis melo L., pests. The geographic areas where they occur overlap, and the same chemicals are used to control both of them. Therefore, to reduce pesticide use, it would be necessary to breed melon lines that simultaneously express a resistance to both insects. Female survival; the time when reproduction starts, peaks, and ends; the number of female offspring at the reproductive peak; and total reproduction (S) were determined under semicontrolled conditions for B. tabaci kept in clip-cages on a susceptible melon genotype Vedrantais, and 12 potential resistant accessions, particularly genotypes expressing the Vat gene controlling resistance to A. gossypii. By using the Lewontin triangular reproductive function and Bootstrapping, the intrinsic rate of increase (r) and its variance were calculated. Statistical analysis showed that the parameter S was as relevant as r for discriminating between the melon accessions. Three genotypes were potential genitors of resistance to the whitefly: PI 161375, PI 414723, and PI 532841. Those possessing the Vat gene were either resistant (PI 161375 and PI 414723) or susceptible (Margot, IsoVat R, and AR 5). This demonstrated the ineffectiveness of Vat against B. tabaci. In this article, we propose a strategy to breed lines that express resistance to aphids and whiteflies on the short-term.