Filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences

Filoviruses are enveloped viruses that cause sporadic outbreaks of severe hemorrhagic fever [CDC, MMWR Morb. Mortal. Wkly. Rep. 50:73-77, 2001; Colebunders and Borchert, J. Infect. 40:16-20, 2000; Colebunders et al., J. Infect. Dis. 196(Suppl. 2):S148-S153, 2007; Geisbert and Jahrling, Nat. Med. 10:S110-S121, 2004]. Previous studies revealed that endosomal cysteine proteases are host factors for ebolavirus Zaire (Chandran et al., Science 308:1643-1645, 2005; Schornberg et al., J. Virol. 80:4174-4178, 2006). In this report, we show that infection mediated by glycoproteins from other phylogenetically diverse filoviruses are also dependent on these proteases and provide additional evidence indicating that they cleave GP1 and expose the binding domain for the critical host factor Niemann-Pick C1. Using selective inhibitors and knockout-derived cell lines, we show that the ebolaviruses Zaire and Cote d'Ivoire are strongly dependent on cathepsin B, while the ebolaviruses Sudan and Reston and Marburg virus are not. Taking advantage of previous studies of cathepsin B inhibitor-resistant viruses (Wong et al., J. Virol. 84:163-175, 2010), we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virus-specific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae.     

UCLA1, a synthetic derivative of a gp120 RNA aptamer, inhibits entry of human immunodeficiency virus type 1 subtype C

Entry of human immunodeficiency virus type 1 (HIV-1) into cells is mediated by the virion surface envelope (Env) glycoproteins, making it a desirable target for antiretroviral entry inhibitors. We previously isolated a family of gp120 binding RNA aptamers and showed that they neutralized the infectivity of HIV-1. In this study, we assessed the activity of a shortened synthetic derivative of the B40 aptamer, called UCLA1, against a large panel of HIV-1 subtype C viruses. UCLA1 tightly bound to a consensus HIV-1 subtype C gp120 and neutralized isolates of the same subtype with 50% inhibitory concentrations (IC(50)s) in the nanomolar range. The aptamer had little toxicity in tests with cell lines and primary cells. Furthermore, it exhibited high therapeutic indices, suggesting that it may be effective at very low doses. Mapping of UCLA1 binding sites on gp120 revealed eight amino acid residues that modulated neutralization resistance. This included residues within the coreceptor binding site, at the base of the V3 loop, and in the bridging sheet within the conserved V1/V2 stem-loop of gp120. The aptamer was also shown to have synergistic effects with T20, a gp41 fusion inhibitor, and IgG1b12 (b12), an anti-CD4 binding site monoclonal antibody. These results suggest that UCLA1 may be suitable for development as a potent HIV-1 entry inhibitor.     

Generation of HIV latency in humanized BLT mice

Here we demonstrate that a combination of tenofovir, emtricitabine, and raltegravir effectively suppresses peripheral and systemic HIV replication in humanized BLT mice. We also demonstrate that antiretroviral therapy (ART)-treated humanized BLT mice harbor latently infected resting human CD4+ T cells that can be induced ex vivo to produce HIV. We observed that the levels of infected resting human CD4+ T cells present in BLT mice are within the range of those observed circulating in patients undergoing suppressive ART. These results demonstrate the potential of humanized BLT mice as an attractive model for testing the in vivo efficacy of novel HIV eradication strategies.     

Differences in kainate receptor involvement in hippocampal mossy fibre long-term potentiation depending on slice orientation

Long-term potentiation (LTP) is a well-established experimental model used to investigate the synaptic basis of learning and memory. LTP at mossy fibre - CA3 synapses in the hippocampus is unusual because it is normally N-methyl-d-aspartate (NMDA) receptor-independent. Instead it seems that the trigger for mossy fibre LTP involves kainate receptors (KARs). Although it is generally accepted that pre-synaptic KARs play an essential role in frequency facilitation and LTP, their subunit composition remains a matter of significant controversy. We have reported previously that both frequency facilitation and LTP can be blocked by selective antagonism of GluK1 (formerly GluR5/Glu(K5))-containing KARs, but other groups have failed to reproduce this effect. Moreover, data from receptor knockout and mRNA expression studies argue against a major role of GluK1, supporting a more central role for GluK2 (formerly GluR6/Glu(K6)). A potential reason underlying the controversy in the pharmacological experiments may reside in differences in the preparations used. Here we show differences in pharmacological sensitivity of synaptic plasticity at mossy fibre - CA3 synapses depend critically on slice orientation. In transverse slices, LTP of fEPSPs was invariably resistant to GluK1-selective antagonists whereas in parasagittal slices LTP was consistently blocked by GluK1-selective antagonists. In addition, there were pronounced differences in the magnitude of frequency facilitation and the sensitivity to the mGlu2/3 receptor agonist DCG-IV. Using anterograde labelling of granule cells we show that slices of both orientations possess intact mossy fibres and both large and small presynaptic boutons. Transverse slices have denser fibre tracts but a smaller proportion of giant mossy fibre boutons. These results further demonstrate a considerable heterogeneity in the functional properties of the mossy fibre projection.     

Ablation of ghrelin receptor in leptin-deficient ob/ob mice has paradoxical effects on glucose homeostasis when compared with ablation of ghrelin in ob/ob mice

The orexigenic hormone ghrelin is important in diabetes because it has an inhibitory effect on insulin secretion. Ghrelin ablation in leptin-deficient ob/ob (Ghrelin(-/-):ob/ob) mice increases insulin secretion and improves hyperglycemia. The physiologically relevant ghrelin receptor is the growth hormone secretagogue receptor (GHS-R), and GHS-R antagonists are thought to be an effective strategy for treating diabetes. However, since some of ghrelin's effects are independent of GHS-R, we have utilized genetic approaches to determine whether ghrelin's effect on insulin secretion is mediated through GHS-R and whether GHS-R antagonism indeed inhibits insulin secretion. We investigated the effects of GHS-R on glucose homeostasis in Ghsr-ablated ob/ob mice (Ghsr(-/-):ob/ob). Ghsr ablation did not rescue the hyperphagia, obesity, or insulin resistance of ob/ob mice. Surprisingly, Ghsr ablation worsened the hyperglycemia, decreased insulin, and impaired glucose tolerance. Consistently, Ghsr ablation in ob/ob mice upregulated negative β-cell regulators (such as UCP-2, SREBP-1c, ChREBP, and MIF-1) and downregulated positive β-cell regulators (such as HIF-1α, FGF-21, and PDX-1) in whole pancreas; this suggests that Ghsr ablation impairs pancreatic β-cell function in leptin deficiency. Of note, Ghsr ablation in ob/ob mice did not affect the islet size; the average islet size of Ghsr(-/-):ob/ob mice is similar to that of ob/ob mice. In summary, because Ghsr ablation in leptin deficiency impairs insulin secretion and worsens hyperglycemia, this suggests that GHS-R antagonists may actually aggravate diabetes under certain conditions. The paradoxical effects of ghrelin ablation and Ghsr ablation in ob/ob mice highlight the complexity of the ghrelin-signaling pathway.     

24-Hour profiles of circulating ghrelin and peptide YY are inversely associated in normal weight premenopausal women

Peptide YY (PYY) and ghrelin (GHR) may modulate one another's actions within the hypothalamus. Peripheral infusion of PYY in humans acutely suppresses circulating concentrations of GHR. Whether an association between PYY and GHR exists in the peripheral circulation of humans over 24 h is unknown. The purpose of this study was to determine if circulating concentrations of PYY and GHR were significantly associated over 24 h in humans. Participants (n = 13) were normal weight, moderately active, women ages 18–24 yr. Blood samples were obtained q10 min for 24 h and assayed using RIA for total PYY and total GHR hourly from 0800 to 1000 h and 2000 to 0800 h and q20 min from 1000 to 2000 h. Dietary intake during the 24 h procedure was comprised of 55% carbohydrates, 30% fat, and 15% protein (three meals and a snack). Statistical analyses included linear mixed-effects modeling to test whether PYY predicted GHR concentrations over 24 h. Participants weighed 57.0 ± 1.5 kg and had 26.1 ± 1.5% body fat (15.0 ± 1.1 kg), 42.1 ± 1.1 kg fat free mass, a BMI of 21.3 ± 0.5 kg/m² and RMR of 1072 ± 28 kcal/24 h. Visually, PYY and GHR exhibited an inverse association over nearly the entire 24 h period. Statistically, circulating concentrations of 24 h PYY predicted 24 h GHR (ghrelin = 1860.51–2.14*PYY; p = 0.04). Circulating concentrations of PYY are inversely associated with GHR over 24 h. These data provide evidence that PYY may contribute to the modulation of the secretion of GHR in normal weight, premenopausal women over a 24 h period and supports similar inferences from experimental studies in animals and humans.     

Apicobasal polarity and cell proliferation during development

Cell polarization and cell division are two fundamental cellular processes. The mechanisms that establish and maintain cell polarity and the mechanisms by which cells progress through the cell cycle are now fairly well understood following decades of experimental work. There is also increasing evidence that the polarization state of a cell affects its proliferative properties. The challenge now is to understand how these two phenomena are mechanistically connected. The aim of the present chapter is to provide an overview of the evidence of cross-talk between apicobasal polarity and proliferation, and the current state of knowledge of the precise mechanism by which this cross-talk is achieved.     

Histopathology confirms white-nose syndrome in bats in Europe

White-nose syndrome, associated with the fungal skin infection geomycosis, caused regional population collapse in bats in North America. Our results, based on histopathology, show the presence of white-nose syndrome in Europe. Dermatohistopathology on two bats (Myotis myotis) found dead in March 2010 with geomycosis in the Czech Republic had characteristics resembling Geomyces destructans infection in bats confirmed with white-nose syndrome in US hibernacula. In addition, a live M. myotis, biopsied for histopathology during hibernation in April 2011, had typical fungal infection with cupping erosion and invasion of muzzle skin diagnostic for white-nose syndrome and conidiospores identical to G. destructans that were genetically confirmed as G. destructans.