A novel circulating tamiami mammarenavirus shows potential for zoonotic spillover

A detailed understanding of the mechanisms underlying the capacity of a virus to break the species barrier is crucial for pathogen surveillance and control. New World (NW) mammarenaviruses constitute a diverse group of rodent-borne pathogens that includes several causative agents of severe viral hemorrhagic fever in humans. The ability of the NW mammarenaviral attachment glycoprotein (GP) to utilize human transferrin receptor 1 (hTfR1) as a primary entry receptor plays a key role in dictating zoonotic potential. The recent isolation of Tacaribe and lymphocytic choriominingitis mammarenaviruses from host-seeking ticks provided evidence for the presence of mammarenaviruses in arthropods, which are established vectors for numerous other viral pathogens. Here, using next generation sequencing to search for other mammarenaviruses in ticks, we identified a novel replication-competent strain of the NW mammarenavirus Tamiami (TAMV-FL), which we found capable of utilizing hTfR1 to enter mammalian cells. During isolation through serial passaging in mammalian immunocompetent cells, the quasispecies of TAMV-FL acquired and enriched mutations leading to the amino acid changes N151K and D156N, within GP. Cell entry studies revealed that both substitutions, N151K and D156N, increased dependence of the virus on hTfR1 and binding to heparan sulfate proteoglycans. Moreover, we show that the substituted residues likely map to the sterically constrained trimeric axis of GP, and facilitate viral fusion at a lower pH, resulting in viral egress from later endosomal compartments. In summary, we identify and characterize a naturally occurring TAMV strain (TAMV-FL) within ticks that is able to utilize hTfR1. The TAMV-FL significantly diverged from previous TAMV isolates, demonstrating that TAMV quasispecies exhibit striking genetic plasticity that may facilitate zoonotic spillover and rapid adaptation to new hosts.     

Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

Anthropogenic changes to leaf litter input affect the fitness of a larval amphibian

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Chronic inflammation and protection from acute hepatitis in transgenic mice expressing TNF in endothelial cells

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-gamma were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.     

Contribution of store-operated Ca2+ entry to pHo-dependent changes in vascular tone of porcine coronary smooth muscle

Vascular smooth muscle contracts on increases of extracellular pH (pH(o)) and relaxes on pH(o) decreases possibly resulting from changes in transsarcolemmal Ca(2+) influx. Therefore, we studied store-operated Ca(2+) entry (SOCE; i.e. capacitative Ca(2+) entry (CCE)) during acidification (pH(o)=6.5) and alkalinization (pH(o)=8.0) in isolated porcine coronary smooth muscle cells (SMCs) by monitoring cytoplasmic Ca(2+) ([Ca(2+)](i)) and divalent cation entry (Mn(2+) quench) with fura-2/AM-fluorometry. Additionally, we evaluated the contribution of SOCE to pH(o)-dependent changes in isometric tension of porcine coronary smooth muscle strips. SOCE elicited in SMCs by the SERCA inhibitor BHQ was strongly modulated by pH(o) showing a decrease upon acidification and vice versa an increase upon alkalinization. BHQ-mediated tension of smooth muscle strips also revealed strong pH(o) dependence. In contrast, L-VOC-dependent tension ([K(+)](o)=20 and 40 mmol l(-1)) was remarkably less affected by pH(o) changes. Moreover, refilling of depleted Ca(2+) stores after repeated M(3)-cholinergic receptor stimulation could be almost completely inhibited by SKF 96365 and was markedly reduced by acidification and considerably enhanced by alkalinization pointing to a major role of SOCE in refilling. We conclude that vascular tone particularly responds to alterations in pH(o) whenever SOCE substantially contributes to the amount of activator Ca(2+) for contraction.     

Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.     

Ecological Effects of Live Salmon Exceed Those of Carcasses During an Annual Spawning Migration

We tested the hypothesis that the carcasses of anadromous Pacific salmon (Oncorhynchus spp.) constitute a significant source of nutrients in the nutrient-poor freshwaters where these fish migrate, spawn, senesce, and die. In a 110 m-long stream reach in Southeast Alaska, we retained nearly 3000 salmon carcasses and compared streamwater nitrogen (N), phosphorus (P), and the biomass of benthic biofilm in this reach with an upstream reference reach. The study spanned 5 months, bracketed the entire salmon run, and encompassed significant seasonal variation in abiotic stream conditions. Concentrations of dissolved and particulate N and P followed distinctly unimodal patterns through time, which tracked the abundance of live salmon, and we observed strong predictive relationships between live-salmon abundance and streamwater-nutrient concentrations. In contrast, we did not observe clear relationships between salmon carcasses and streamwater nutrients. Biofilm biomass within our study reaches seemed to more closely track the abundance of live salmon than the abundance of carcasses. The experimental retention of carcasses had a minor or undetectable influence on nutrient concentrations and biofilm within the study reach as compared to the reference reach. We conclude that physical factors such as temperature, discharge, nutrient limitation, and irradiance vary seasonally in ways that maximize the influence of nutrients provisioned by live salmon and minimize the influence of carcass-derived nutrients on the aspects of stream ecosystems that we examined. Overall, our results promote a new perspective on the ecological role of salmon in freshwaters, and contribute to a more mechanistic understanding of how migratory fishes can influence aquatic ecosystems.