Herpes Simplex Virus Type 1 Glycoprotein E Mediates Retrograde Spread from Epithelial Cells to Neurites

In animal models of infection, glycoprotein E (gE) is required for efficient herpes simplex virus type 1 (HSV-1) spread from the inoculation site to the cell bodies of innervating neurons (retrograde direction). Retrograde spread in vivo is a multistep process, in that HSV-1 first spreads between epithelial cells at the inoculation site, then infects neurites, and finally travels by retrograde axonal transport to the neuron cell body. To better understand the role of gE in retrograde spread, we used a compartmentalized neuron culture system, in which neurons were infected in the presence or absence of epithelial cells. We found that gE-deleted HSV-1 (NS-gEnull) retained retrograde axonal transport activity when added directly to neurites, in contrast to the retrograde spread defect of this virus in animals. To better mimic the in vivo milieu, we overlaid neurites with epithelial cells prior to infection. In this modified system, virus infects epithelial cells and then spreads to neurites, revealing a 100-fold retrograde spread defect for NS-gEnull. We measured the retrograde spread defect of NS-gEnull from a variety of epithelial cell lines and found that the magnitude of the spread defect from epithelial cells to neurons correlated with epithelial cell plaque size defect, indicating that gE plays a similar role in both types of spread. Therefore, gE-mediated spread between epithelial cells and neurites likely explains the retrograde spread defect of gE-deleted HSV-1 in vivo.Herpes simplex virus type 1 (HSV-1) is an alphaherpesvirus that characteristically infects skin and mucosal surfaces before spreading to sensory neurons, where it establishes a lifelong persistent infection. The virus periodically returns to the periphery via sensory axons and causes recurrent lesions as well as asymptomatic shedding. This life cycle requires viral transport along axons in two directions: toward the neuron cell body (retrograde direction) and away from the neuron cell body (anterograde direction).Many studies of alphaherpesvirus neuronal spread have focused on pseudorabies virus (PRV), a virus whose natural host is the pig. Three PRV proteins, glycoprotein E (gE), gI, and Us9, have been shown to mediate anterograde neuronal spread both in animal models of infection and in cultured neurons. However, these three proteins are dispensable for retrograde spread (3, 8, 11, 12, 31, 46). In contrast, numerous animal models of infection have shown that HSV-1 gE is required for retrograde spread from the inoculation site to the cell bodies of innervating neurons (4, 9, 44, 56). In the murine flank model, wild-type (WT) virus replicates in the skin and then infects sensory neurons and spreads in a retrograde direction to the dorsal root ganglia (DRG). In this model, gE-deleted HSV-1 replicates in the skin but is not detected in the DRG (9, 44). This phenotype differs from gE-deleted PRV, which is able to reach the DRG at WT levels (8). Thus, unlike PRV, gE-deleted HSV-1 viruses have a retrograde spread defect in vivo.HSV-1 gE is a 552-amino-acid type I membrane protein found in the virion membrane as well as in the trans-Golgi and plasma membranes of infected cells (1). gE forms a heterodimer with another viral glycoprotein, gI. The gE/gI complex is important for HSV-1 immune evasion through its Fc receptor activity. gE/gI binds to the Fc domain of antibodies directed against other viral proteins, sequestering these antibodies and blocking antibody effector functions (27, 32, 40). Additionally, gE/gI promotes spread between epithelial cells. Viruses lacking either gE or gI form characteristically small plaques in cell culture and small inoculation site lesions in mice (4, 9, 18, 40, 58). In animal models, gE and gI also mediate viral spread in both anterograde and retrograde directions (4, 19, 44, 56).In order to better understand the role of gE in HSV-1 retrograde neuronal spread, we employed a compartmentalized neuron culture system that has been used to study directional neuronal spread of PRV and West Nile virus (12, 14, 45). In the Campenot chamber system, neurites are contained in a compartment that is separate from their corresponding cell bodies. Therefore, spread in an exclusively retrograde direction can be measured by infecting neurites and detecting spread to neuron cell bodies.HSV-1 replication requires retrograde transport of incoming viral genomes to the nucleus. In neurites, fusion between viral and cellular membranes occurs at the plasma membrane (43, 48). Upon membrane fusion, the capsid and a subset of tegument proteins (the inner tegument) dissociate from glycoproteins and outer tegument proteins, which remain at the plasma membrane (28, 38). Unenveloped capsids and the associated inner tegument proteins are then transported in the retrograde direction to the nucleus (7, 48, 49).For both neurons and epithelial cells, retrograde transport is dependent upon microtubules, ATP, the retrograde microtubule motor dynein, and the dynein cofactor dynactin (22, 34, 49, 52). Several viral proteins interact with components of the dynein motor complex (23, 39, 60). However, none of these proteins suggest a completely satisfactory mechanism by which viral retrograde transport occurs, either because they are not components of the complex that is transported to the nucleus (UL34, UL9, VP11/12) or because capsids lacking that protein retain retrograde transport activity (VP26) (2, 17, 21, 28, 37). This implies that additional viral proteins are involved in retrograde trafficking.We sought to better characterize the role of gE in retrograde spread and found that gE is dispensable for retrograde axonal transport; however, it promotes HSV-1 spread from epithelial cells to neurites. This epithelial cell-to-neuron spread defect provides a plausible explanation for the retrograde spread defect of gE-deleted HSV-1 in animal models of infection.     

Consequences of constitutive and induced variation in plant nutritional quality for immune defence of a herbivore against parasitism

The mechanisms through which trophic interactions between species are indirectly mediated by distant members in a food web have received increasing attention in the field of ecology of multitrophic interactions. Scarcely studied aspects include the effects of varying plant chemistry on herbivore immune defences against parasitoids. We investigated the effects of constitutive and herbivore-induced variation in the nutritional quality of wild and cultivated populations of cabbage (Brassica oleracea) on the ability of small cabbage white Pieris rapae (Lepidoptera, Pieridae) larvae to encapsulate eggs of the parasitoid Cotesia glomerata (Hymenoptera, Braconidae). Average encapsulation rates in caterpillars parasitised as first instars were low and did not differ among plant populations, with caterpillar weight positively correlating with the rates of encapsulation. When caterpillars were parasitised as second instar larvae, encapsulation of eggs increased. Caterpillars were larger on the cultivated Brussels sprouts plants and exhibited higher levels of encapsulation compared with caterpillars on plants of either of the wild cabbage populations. Observed differences in encapsulation rates between plant populations could not be explained exclusively by differences in host growth on the different Brassica populations. Previous herbivore damage resulted in a reduction in the larval weight of subsequent herbivores with a concomitant reduction in encapsulation responses on both Brussels sprouts and wild cabbage plants. To our knowledge this is the first study demonstrating that constitutive and herbivore-induced changes in plant chemistry act in concert, affecting the immune response of herbivores to parasitism. We argue that plant-mediated immune responses of herbivores may be important in the evaluation of fitness costs and benefits of herbivore diet on the third trophic level.     

Discovery of novel aminothiadiazole amides as selective EP3 receptor antagonists

This Letter discloses a series of 2-aminothiadiazole amides as selective EP₃ receptor antagonists. SAR optimization resulted in compounds with excellent functional activity in vitro. In addition, efforts to optimize DMPK properties in the rat are discussed. These efforts have resulted in the identification of potent, selective EP₃ receptor antagonists with excellent DMPK properties suitable for in vivo studies.     

Rescue of ��F508-CFTR by the SGK1/Nedd4-2 Signaling Pathway

The most common mutation in cystic fibrosis (CF) is ΔF508, which is associated with failure of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) to traffic to the plasma membrane. By a still unknown mechanism, the loss of correctly trafficked ΔF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane. ENaC trafficking is known to be regulated by a signaling pathway involving the glucocorticoid receptor, the serum- and glucocorticoid-regulated kinase SGK1, and the ubiquitin E3 ligase Nedd4-2. We show here that dexamethasone rescues functional expression of ΔF508-CFTR. The half-life of ΔF508-CFTR is also dramatically enhanced. Dexamethasone-activated ΔF508-CFTR rescue is blocked either by the glucocorticoid receptor antagonist RU38486 or by the phosphatidylinositol 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Co-immunoprecipitation studies indicate that Nedd4-2 binds to both wild-type- and ΔF508-CFTR. These complexes are inhibited by dexamethasone treatment, and CFTR ubiquitination is concomitantly decreased. We further show that knockdown of Nedd4-2 by small interfering RNA also corrects ΔF508-CFTR trafficking. Conversely, knockdown of SGK1 by small interfering RNA completely blocks dexamethasone-activated ΔF508-CFTR rescue. These data suggest that the SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells.Cystic fibrosis (CF)² is the most common life-limiting genetic disease in the United States and is due to mutations in the CFTR gene. The most common mutation, ΔF508-CFTR, results in a failure of the mutant protein to traffic properly to the apical plasma membrane of epithelial cells in the lung and other organs (1, 2). By a still unknown mechanism, the loss of correctly trafficked ΔF508-CFTR results in an excess of the epithelial sodium channel (ENaC) on the apical plasma membrane (3–5). In the CF lung, such high levels of ENaC activity are believed to cause dehydration of the airway, and the consequent proinflammatory condition that characterizes CF lung pathophysiology. Similar proinflammatory pathophysiology has been reported to characterize the lung of transgenic mice which overexpress β-ENaC (6). Operationally, it seems that when membrane-localized CFTR decreases in CF, ENaC activity at the plasma membrane increases; CF-related morbidity and mortality follow.In the case of ENaC trafficking, the process is known to be regulated by a glucocorticoid receptor/SGK1 signaling pathway affecting phosphorylation of the ubiquitin ligase E3 protein Nedd4-2 (7, 8). Fig. 1 illustrates how surface expression of ENaC is controlled by the serum- and glucocorticoid-inducible kinase SGK1, the upstream signal, and the ubiquitin E3 ligase Nedd4-2, the downstream signal. Under default conditions, Nedd4-2 suppresses ENaC surface expression by binding to ENaC via the interaction between the PPXY motifs of ENaC and WW domains on Nedd4-2. Nedd4-2 then catalyzes the ubiquitination of bound ENaC. This step targets ENaC for proteasomal degradation (9, 10). However, when Nedd4-2 is phosphorylated by SGK1, the default interaction between Nedd4-2 and ENaC is reduced, and ENaC is maintained at the plasma membrane (7, 8). The requirement for Nedd4-2 for destruction of ENaC is supported by the recent observation that siRNA against Nedd4-2 is sufficient to permit ENaC to be expressed at the plasma membrane (10). Importantly, both glucocorticoid receptor (GR) and phosphoinositide-3-kinase (PI 3-kinase) signaling pathways must be present for high levels of Na⁺ transport to occur. For example, treatment with the GR antagonist RU38486 (11–13) or the PI 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (14–16) results in a complete loss of glucocorticoid-activated ENaC activity.Open in a separate windowFIGURE 1.Schematic diagram of regulation of ENaC and CFTR by SGK1/Nedd4-2. The surface expression of ENaC is controlled by the serum/glucocorticoid inducible kinase SGK1, the upstream signal, and the neural precursor cell-expressed developmentally down-regulated isoform 2 (Nedd4-2), the downstream signal. The solid black arrows trace the signal to a point where phospho-Nedd4-2 releases ENaC, thereby saving it from default ubiquitination and proteasomal destruction. ENaC is then maintained at the plasma membrane. Glucocorticoid-activated ENaC membrane trafficking is blocked by the glucocorticoid receptor antagonist RU38486 and the PI 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Alternatively, silencing of endogenous Nedd4-2 by siRNA enhances ENaC trafficking to the plasma membrane. (+) indicates positive regulation, and (−) indicates negative regulation.The placement of the parenthetical (CFTR) in the SGK1/Nedd4-2 signaling pathway (Fig. 1) serves to underscore our hypothesis that CFTR itself could play an interactive or parallel role in the SGK1/Nedd4-2/ENaC-trafficking mechanism. This hypothesis seems reasonable because the regulatory effects of SGK1 and Nedd4-2 are not limited to trafficking of ENaC but also regulate several other epithelial channels and transporters (17, 18). Additionally, co-expression studies in Xenopus oocytes (19, 20) have shown that SGK1 appears to greatly enhance the functional activity of CFTR.In this report we have shown that activation of the SGK1 signaling pathway by the glucocorticoid dexamethasone results in the rescue of ΔF508-CFTR. The half-life of ΔF508-CFTR, once it reaches the plasma membrane, is also dramatically enhanced. Consistently, glucocorticoid-activated ΔF508-CFTR rescue is blocked by the GR antagonist RU38486 and by the PI 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 as well as by knockdown of endogenous SGK1 by siRNA. We have further shown that at the downstream end of the SGK1/Nedd4-2 signaling pathway, knockdown of Nedd4-2 by siRNA also results in ΔF508-CFTR rescue. Finally, co-immunoprecipitation studies indicated that Nedd4-2 binds to both WT- and ΔF508-CFTR and that treatment with either glucocorticoid or Nedd4-2 siRNA reduces formation of Nedd4-2·CFTR complexes as well as ubiquitination of ΔF508-CFTR. Consistently, chloride transport is well correlated with the level of plasma membrane expression of ΔF508-CFTR protein. These data suggest that the glucocorticoid receptor-dependent SGK1/Nedd4-2 signaling pathway regulates both CFTR and ENaC trafficking in CF epithelial cells. We interpret these results to indicate that drugs affecting the SGK1/Nedd4-2 signaling pathway may be promising targets for cystic fibrosis therapeutic development.     

Composition and significance of picophytoplankton in Antarctic waters

Filter fractionated picophytoplankton from Antarctic coastal waters (summer 2001) represented only 7–33% of total phytoplankton, even though total stocks were low (average Chl a = 0.32 μg l⁻¹, range = 0.13–1.03 μg l⁻¹). Though all cells passed a 2 μm filter, electron microscopy revealed most cells were over 2 μm, principally Parmales, Phaeocystis sp., and small diatoms. CHEMTAX analysis of HPLC pigment data suggested type 8 haptophytes (e.g. Phaeocystis sp. plus Parmales and pelagophytes) contributed 7–58% of picoplanktonic chlorophyll a, type 6 haptophytes (e.g. coccolithophorids) 18–59%, diatoms 0–18% (mostly type 2 diatoms, e.g. Pseudonitzschia sp., 0–15%), prasinophytes 0–17%, with cell fragments of cryptophytes 0–40%, and dinoflagellates 0–11%. Only stocks of type 8 haptophytes and prasinophytes differed significantly due to successional changes. Zeaxanthin concentrations exceeded estimates from previous cyanobacterial counts and may derive from non-photosynthetic bacteria.     

A captive feeding study with the Pacific harbor seal (Phoca vitulina richardii): Implications for scat analysis

Seven prey species ( n _total > 2,700) were fed to seven captive male Pacific harbor seals ( Phoca vitulina richardii ) in 177 experimental meals to quantify biases associated with scat analysis and current consumption models. Hard parts from an individual meal were recovered in an average of 3.8 ± 1.8 scats (range 1–10; mean ± SD). Overall, 57.7 ± 33.2% of otoliths and 89.5 ± 15.5% of squid beaks were recovered. Recovery rates varied, and prey with smaller, fragile otoliths were recovered in lesser quantities than prey with larger, robust otoliths. Recovery rates of all prey except pink salmon were improved by a mean of 31.7% when all diagnostic structures were included in estimates. Estimated recovery of pink salmon was 9.5 times that fed seals based on the all-structure technique. Mean length reduction of recovered otoliths was 20.4 ± 10.1%. Correction factors calculated from average length reduction improved length estimates for all fish species. Grade-specific length correction factors (gLCFs) reduced variability in all of the estimates and significantly improved estimates of prey with highly eroded otoliths including Pacific hake and shortbelly rockfish. The Biomass Reconstruction (BR) model accurately predicted biomass consumption within 4% of known consumption, whereas estimates based on frequency of occurrence were inaccurate.