Lipid Raft- and Src Family Kinase-Dependent Entry of Coxsackievirus B into Human Placental Trophoblasts

Maternal-fetal transmission of group B coxsackieviruses (CVB) during pregnancy has been associated with a number of diverse pathological outcomes, including hydrops fetalis, fetal myocarditis, meningoencephalitis, neurodevelopmental delays, congenital skin lesions, miscarriage, and/or stillbirth. Throughout pregnancy, the placenta forms a critical antimicrobial protective barrier at the maternal-fetal interface. Despite the severity of diseases accompanying fetal CVB infections, little is known regarding the strategies used by CVB to gain entry into placental trophoblasts. Here we used both a trophoblast cell line and primary human trophoblasts to demonstrate the mechanism by which CVB gains entry into polarized placental trophoblasts. Our studies revealed that the kinetics of CVB entry into placental trophoblasts are similar to those previously described for polarized intestinal epithelial cells. Likewise, CVB entry into placental trophoblasts requires decay-accelerating factor (DAF) binding and involves relocalization of the virus from the apical surface to intercellular tight junctions. In contrast, we have identified a divergent mechanism for CVB entry into polarized trophoblasts that is clathrin, caveolin-1, and dynamin II independent but requires intact lipid rafts. In addition, we found that members of the Src family of tyrosine kinases were required for CVB entry. Our studies highlight the complexity of viral entry into human placental trophoblasts and may serve as a model for mechanisms used by diverse pathogens to penetrate the placental barrier.     

Calcium signals and calpain-dependent necrosis are essential for release of coxsackievirus B from polarized intestinal epithelial cells

Coxsackievirus B (CVB), a member of the enterovirus family, targets the polarized epithelial cells lining the intestinal tract early in infection. Although the polarized epithelium functions as a protective barrier, this barrier is likely exploited by CVB to promote viral entry and subsequent egress. Here we show that, in contrast to nonpolarized cells, CVB-infected polarized intestinal Caco-2 cells undergo nonapoptotic necrotic cell death triggered by inositol 1,4,5-trisphosphate receptor-dependent calcium release. We further show that CVB-induced cellular necrosis depends on the Ca(2+)-activated protease calpain-2 and that this protease is involved in CVB-induced disruption of the junctional complex and rearrangements of the actin cytoskeleton. Our study illustrates the cell signaling pathways hijacked by CVB, and perhaps other viral pathogens, to promote their replication and spread in polarized cell types.     

Modulation of venular microvessel permeability by calcium influx into endothelial cells.

It has been proposed that calcium ion influx into endothelial cells modulates the permeability of venular microvessels via a calcium-dependent contractile process. The results of recent investigations using permeabilized endothelial cell monolayers conform to this hypothesis by demonstrating a calcium-dependent interaction of endothelial actin and myosin during the retraction of adjacent endothelial cells exposed to inflammatory agents. Little is known about the pathway for calcium influx into endothelial cells after exposure to mediators of inflammation, but evidence suggests that the properties of the calcium entry pathways are similar to the calcium entry pathways that regulate the release of endothelium-derived relaxing factor (EDRF). Substances that stimulate EDRF release from arterial endothelium also increase venular microvessel permeability. Recently developed methods to measure cytoplasmic calcium concentration in the endothelial cells forming the walls of individually perfused microvessels enable a direct investigation of the modulation of the permeability of venular microvessels by calcium influx. These experiments demonstrate that the magnitude of the initial increase in the permeability of microvessels after exposure to an agent that increases permeability, such as a calcium ionophore, is determined by the magnitude of calcium ion influx into the endothelial cells. Furthermore, the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential of the endothelial cells. Depolarization of the endothelial cell membrane reduces calcium influx and attenuates increases in permeability whereas hyperpolarization of the endothelial membrane increases calcium influx and potentiates increases in permeability. These data conform to the hypothesis that a passive conductance channel for calcium is a major pathway for calcium ion flux responsible to eliciting an increase in the permeability of the endothelial barrier in microvessels.     

Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5

The major group B coxsackievirus (CVB) receptor is a component of the epithelial tight junction (TJ), a protein complex that regulates the selective passage of ions and molecules across the epithelium. CVB enters polarized epithelial cells from the TJ, causing a transient disruption of TJ integrity. Here we show that CVB does not induce major reorganization of the TJ, but stimulates the specific internalization of occludin-a TJ integral membrane component-within macropinosomes. Although occludin does not interact directly with virus, depletion of occludin prevents CVB entry into the cytoplasm and inhibits infection. Both occludin internalization and CVB entry require caveolin but not dynamin; both are blocked by inhibitors of macropinocytosis and require the activity of Rab34, Ras, and Rab5, GTPases known to regulate macropinocytosis. Thus, CVB entry depends on occludin and occurs by a process that combines aspects of caveolar endocytosis with features characteristic of macropinocytosis.     

Hantavirus causing hemorrhagic fever with renal syndrome enters from the apical surface and requires decay-accelerating factor (DAF/CD55)

The Old World hantaviruses, members of the family Bunyaviridae, cause hemorrhagic fever with renal syndrome (HFRS). Transmission to humans occurs via inhalation of aerosols contaminated with the excreta of infected rodents. The viral antigen is detectable in dendritic cells, macrophages, lymphocytes, and, most importantly, microvascular endothelial cells. However, the site and detailed mechanism of entry of HFRS-causing hantaviruses in polarized epithelial cells have not yet been defined. Therefore, this study focused on the entry of the pathogenic hantaviruses Hantaan and Puumala into African green monkey kidney epithelial cells and primary human endothelial cells. The polarized epithelial and endothelial cells were found to be susceptible to hantavirus infection exclusively from the apical surface. Treatment with phosphatidylinositol-specific phospholipase C, which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface, protects cells from infection, indicating that hantaviruses require a GPI-anchored protein as a cofactor for entry. Decay-accelerating factor (DAF)/CD55 is a GPI-anchored protein of the complement regulatory system and serves as a receptor for attachment to the apical cell surface for a number of viruses. Infection was reduced by the pretreatment of hantaviral particles with human recombinant DAF. Moreover, the treatment of permissive cells with DAF-specific antibody blocked infection. These results demonstrate that the Old World hantaviruses Hantaan and Puumala enter polarized target cells from the apical site and that DAF is a critical cofactor for infection.     

Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos

Using calcium-sensitive dyes together with their dextran conjugates and confocal microscopy, we have looked for evidence of localized calcium signaling in the region of the nucleus before entry into mitosis, using the sea urchin egg first mitotic cell cycle as a model. Global calcium transients that appear to originate from the nuclear area are often observed just before nuclear envelope breakdown (NEB). In the absence of global increases in calcium, confocal microscopy using Calcium Green- 1 dextran indicator dye revealed localized calcium transients in the perinuclear region. We have also used a photoinactivatable calcium chelator, nitrophenyl EGTA (NP-EGTA), to test whether the chelator- induced block of mitosis entry can be reversed after inactivation of the chelator. Cells arrested before NEB by injection of NP-EGTA resume the cell cycle after flash photolysis of the chelator. Photolysis of chelator triggers calcium release. TreatmenT with caFfeine to enhance calcium-induced calcium release increases the amplitude of NEB- associated calcium transients. These results indicate that calcium increases local to the nucleus are required to trigger entry into mitosis. Local calcium transients arise in the perinuclear region and can spread from this region into the cytoplasm. Thus, cell cycle calcium signals are generated by the perinuclear mitotic machinery in early sea urchin embryos.     

Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores

Calcium-mediated microneme secretion in Toxoplasma gondii is stimulated by contact with host cells, resulting in the discharge of adhesins that mediate attachment. The intracellular source of calcium and the signaling pathway(s) triggering release have not been characterized, prompting our search for mediators of calcium signaling and microneme secretion in T. gondii. We identified two stimuli of microneme secretion, ryanodine and caffeine, which enhanced release of calcium from parasite intracellular stores. Ethanol, a previously characterized trigger of microneme secretion, stimulated an increase in parasite inositol 1,4,5-triphosphate, implying that this second messenger may mediate intracellular calcium release. Consistent with this observation, xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, inhibited microneme secretion and blocked parasite attachment and invasion of host cells. Collectively, these results suggest that T. gondii possess an intracellular calcium release channel with properties of the inositol 1,4,5-triphosphate/ryanodine receptor superfamily. Intracellular calcium channels, previously studied almost exclusively in multicellular animals, appear to also be critical to the control of parasite calcium during the initial steps of host cell entry.     

Store-operated calcium entry and increased endothelial cell permeability

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.     

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.     

Heparan sulfates and coxsackievirus-adenovirus receptor: each one mediates coxsackievirus B3 PD infection

Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.     

Pharmacology of capacitative calcium entry

The versatility of Ca(2+) as a messenger in multiple signaling events requires that the concentration of calcium ions within the cytoplasm be highly regulated. In particular, the release of calcium from intracellular stores must often be linked to calcium influx across the cell membrane. Capacitative calcium entry, whereby the depletion of intracellular Ca(2+) stores induces the influx of extracellular calcium, is a crucial element of concerted calcium signaling. Investigations into the phenomenon are contributing to a new appreciation for the organized cytoplasmic framework that supports calcium signaling.     

Multiple receptor interactions trigger release of membrane and intracellular calcium stores critical for herpes simplex virus entry

Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. Confocal microscopy and small interfering RNA (siRNA) were used to identify the source of the calcium and to dissect the requisite viral-cell interactions. Binding of HSV to human epithelial cells induced no calcium response, but shifting the cells to temperatures permissive for penetration triggered increases in plasma membrane calcium followed by a global release of intracellular calcium. Transfection with siRNA targeting the proteoglycan syndecan-2 blocked viral binding and abrogated any calcium response. Transfection with siRNA targeting nectin-1, a glycoprotein D receptor, also prevented both membrane and intracellular calcium responses. In contrast, the membrane response was preserved after transfection with siRNA targeting integrinalphav, a novel glycoprotein H receptor. The membrane response, however, was not sufficient for viral entry, which required interactions with integrinalphav and release of inositol-triphosphate receptor-dependent intracellular calcium stores. Thus, calcium plays a critical, complex role in HSV entry.     

Genetic evidence supporting caveolae microdomain regulation of calcium entry in endothelial cells

Various cellular signals initiate calcium entry into cells, and there is evidence that lipid rafts and caveolae may concentrate proteins that regulate transmembrane calcium fluxes. Here, using mice deficient in caveolin-1 (Cav-1) and Cav-1 knock-out reconstituted with endothelium-specific Cav-1, we show that Cav-1 is essential for calcium entry in endothelial cells and governs the localization and protein-protein interactions between transient receptor channels C4 and C1. Thus, Cav-1 is required for calcium entry in vascular endothelial cells and perhaps other specialized cell types containing caveolae.     

Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions

Group B coxsackieviruses (CVBs) must cross the epithelium as they initiate infection, but the mechanism by which this occurs remains uncertain. The coxsackievirus and adenovirus receptor (CAR) is a component of the tight junction and is inaccessible to virus approaching from the apical surface. Many CVBs also interact with the GPI-anchored protein decay-accelerating factor (DAF). Here, we report that virus attachment to DAF on the apical cell surface activates Abl kinase, triggering Rac-dependent actin rearrangements that permit virus movement to the tight junction. Within the junction, interaction with CAR promotes conformational changes in the virus capsid that are essential for virus entry and release of viral RNA. Interaction with DAF also activates Fyn kinase, an event that is required for the phosphorylation of caveolin and transport of virus into the cell within caveolar vesicles. CVBs thus exploit DAF-mediated signaling pathways to surmount the epithelial barrier.     

Calcium–Permeable Channels and Endothelial Dysfunction in Acute Lung Injury

The increased permeability of the lung microvascular endothelium is one critical initiation of acute lung injury (ALI). The disruption of vascular-endothelium integrity results in leakiness of the endothelial barrier and accumulation of protein-rich fluid in the alveoli. During ALI, increased endothelial-cell (EC) permeability is always companied by high frequency and amplitude of cytosolic Ca²⁺ oscillations. Mechanistically, cytosolic calcium oscillations include calcium release from internal stores and calcium entry via channels located in the cell membrane. Recently, numerous publications have shown substantial evidence that calcium-permeable channels play an important role in maintaining the integrity of the endothelium barrier function of the vessel wall in ALI. These novel endothelial signaling pathways are future targets for the treatment of lung injury. This short review focuses on the up-to-date research and provide insight into the contribution of calcium influx via ion channels to the disruption of lung microvascular endothelial-barrier function during ALI.     

Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent

The role of signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) during viral infection has gained much recent attention. Our laboratory reported on an important regulatory role for extracellular signal-regulated kinases (ERK1/2), subfamily members of the MAPKs, during coxsackievirus B3 (CVB3) infection. However, the role of the PI3K pathway in CVB3 infection has not been well characterized. CVB3 is the most common known viral infectant of heart muscle that directly injures and kills infected cardiac myocytes during the myocarditic process. In the present study, we investigated the role of protein kinase B (PKB) (also known as Akt), a general downstream mediator of survival signals through the PI3K cascade, in regulating CVB3 replication and virus-induced apoptosis in a well-established HeLa cell model. We have demonstrated that CVB3 infection leads to phosphorylation of PKB/Akt on both Ser-473 and Thr-308 residues through a PI3K-dependent mechanism. Transfection of HeLa cells with a dominant negative mutant of Akt1 or pretreatment of wild-type HeLa cells with the specific PI3K inhibitor LY294002 significantly suppresses viral RNA expression, as reflected in diminished viral capsid protein expression and viral release. Dominant negative Akt1 and LY294002 also increase apoptosis in infected cells, which can be reversed by addition of the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Interestingly, blocking of apoptosis by zVAD.fmk does not reverse the viral RNA translation blockade, indicating that the inhibitory effect of dominant negative Akt1 on viral protein expression is not caspase dependent. In addition, we showed that the attachment of virus to its receptor-coreceptor complex is not sufficient for PKB/Akt activation and that postentry viral replication is required for Akt phosphorylation. Taken together, these data illustrate a new and imperative role for Akt in CVB3 infection in HeLa cells and show that the PI3K/Akt signaling is beneficial to CVB3 replication.     

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH(2) and SLIGKV-NH(2,) respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.     

Opioid Receptor Regulation of Neuronal Voltage-Gated Calcium Channels

Neuronal voltage-gated calcium channels play a pivotal role in the conversion of electrical signals into calcium entry into nerve endings that is required for the release of neurotransmitters. They are under the control of a number of cellular signaling pathways that serve to fine tune synaptic activities, including G-protein coupled receptors (GPCRs) and the opioid system. Besides modulating channel activity via activation of second messengers, GPCRs also physically associate with calcium channels to regulate their function and expression at the plasma membrane. In this mini review, we discuss the mechanisms by which calcium channels are regulated by classical opioid and nociceptin receptors. We highlight the importance of this regulation in the control of neuronal functions and their implication in the development of disease conditions. Finally, we present recent literature concerning the use of novel μ-opioid receptor/nociceptin receptor modulators and discuss their use as potential drug candidates for the treatment of pain.

     

Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections

A subset of human papillomavirus (HPV) infections is causally related to the development of human epithelial tumors and cancers. Like a number of pathogens, HPV entry into target cells is initiated by first binding to heparan sulfonated proteoglycan (HSPG) cell surface attachment factors. The virus must then move to distinct secondary receptors, which are responsible for particle internalization. Despite intensive investigation, the mechanism of HPV movement to and the nature of the secondary receptors have been unclear. We report that HPV16 particles are not liberated from bound HSPG attachment factors by dissociation, but rather are released by a process previously unreported for pathogen-host cell interactions. Virus particles reside in infectious soluble high molecular weight complexes with HSPG, including syndecan-1 and bioactive compounds, like growth factors. Matrix mellatoproteinase inhibitors that block HSPG and virus release from cells interfere with virus infection. Employing a co-culture assay, we demonstrate HPV associated with soluble HSPG-growth factor complexes can infect cells lacking HSPG. Interaction of HPV-HSPG-growth factor complexes with growth factor receptors leads to rapid activation of signaling pathways important for infection, whereas a variety of growth factor receptor inhibitors impede virus-induced signaling and infection. Depletion of syndecan-1 or epidermal growth factor and removal of serum factors reduce infection, while replenishment of growth factors restores infection. Our findings support an infection model whereby HPV usurps normal host mechanisms for presenting growth factors to cells via soluble HSPG complexes as a novel method for interacting with entry receptors independent of direct virus-cell receptor interactions.