Infection Of Human Endothelial Cells Research Articles

Infection of Human Endothelial Cells with Lassa Virus Induces Early but Transient Activation and Low Type I IFN Response Compared to the Closely-Related Nonpathogenic Mopeia Virus

Lassa virus (LASV), an Old World arenavirus, is responsible for hemorrhagic fevers in western Africa. The privileged tropism of LASV for endothelial cells combined with a dysregulated inflammatory response are the main cause of the increase in vascular permeability observed during the disease. Mopeia virus (MOPV) is another arenavirus closely related to LASV but nonpathogenic for non-human primates (NHPs) and has never been described in humans. MOPV is more immunogenic than LASV in NHPs and in vitro in human immune cell models, with more intense type I IFN and adaptive cellular responses. Here, we compared the transcriptomic and proteomic responses of human umbilical vein endothelial cells (HUVECs) to infection with the two viruses to further decipher the mechanisms involved in their differences in immunogenicity and pathogenicity. Both viruses replicated durably and efficiently in HUVECs, but the responses they induced were strikingly different. Modest activation was observed at an early stage of LASV infection and then rapidly shut down. By contrast, MOPV induced a late but more intense response, characterized by the expression of genes and proteins mainly associated with the type I IFN response and antigen processing/presentation. Such a response is consistent with the higher immunogenicity of MOPV relative to LASV, whereas the lack of an innate response induced in HUVECs by LASV is consistent with its uncontrolled systemic dissemination through the vascular endothelium.

Extracellular vimentin is an attachment factor that facilitates SARS-CoV-2 entry into human endothelial cells

SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography-tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

A CRISPR-Cas9 screen identifies mitochondrial translation as an essential process in latent KSHV infection of human endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). The main proliferating component of KS tumors is a cell of endothelial origin termed the spindle cell. Spindle cells are predominantly latently infected with only a small percentage of cells undergoing viral replication. As there is no direct treatment for latent KSHV, identification of host vulnerabilities in latently infected endothelial cells could be exploited to inhibit KSHV-associated tumor cells. Using a pooled CRISPR-Cas9 lentivirus library, we identified host factors that are essential for the survival or proliferation of latently infected endothelial cells in culture, but not their uninfected counterparts. Among the many host genes identified, there was an enrichment in genes localizing to the mitochondria, including genes involved in mitochondrial translation. Antibiotics that inhibit bacterial and mitochondrial translation specifically inhibited the expansion of latently infected endothelial cells and led to increased cell death in patient-derived PEL cell lines. Direct inhibition of mitochondrial respiration or ablation of mitochondrial genomes leads to increased death in latently infected cells. KSHV latent infection decreases mitochondrial numbers, but there are increases in mitochondrial size, genome copy number, and transcript levels. We found that multiple gene products of the latent locus localize to the mitochondria. During latent infection, KSHV significantly alters mitochondrial biology, leading to enhanced sensitivity to inhibition of mitochondrial respiration, which provides a potential therapeutic avenue for KSHV-associated cancers.

Pathogenic Mechanisms of Endothelial Damage in Severe Scrub Typhus

Abstract Our newly established lethal and sublethal Orientia tsutsugamushi infection mouse models, which mimic clinical/pathologic features of human scrub typhus, are valuable for mechanistic studies. A hallmark of lethal Orientia infection is Th1-skewed, but Th2-impared, immune responses, accompanied with severe endothelial damage and acute tissue injury. Importantly, while tissue bacteria peak at disease onset (D6) in mice, neutrophil and CD8 T cell influx/activation reach peaks in the lungs around D10 (prior to host death). We hypothesize a two-stage-dysfunction loop for lethal infection: 1) Orientia replication in phagocytes and endothelial cells (EC) triggers inflammatory responses and host defense machinery; and 2) infection-mediated release of DAMP molecules exacerbates vascular dysfunction. This hypothesis was supported by immunofluorescent co-staining of bacteria with leukocyte/EC/platelet-specific markers, multi-color FACS, Western blot, and qRT-PCR. The progressive loss of vasculature and junction proteins (VE-cad, occludin) and reduction in vascular function (angiopoietin 1, Tie2, pTie2) and CD41+ platelets in the lungs were positively linked to neutrophil and CD8 T cell influx and activation, rather than to body bacterial loads. The mechanisms of immune dysregulation were further examined by using infected human endothelial cells and mouse primary macrophage and neutrophil cultures, as well as knockout mice. While Orientia infection directly altered the Ang-Tie2 axis in EC, activated IL-33/IL-36-like DAMP molecules further contributed to vascular/tissue injury. Our studies, for the first time, have revealed immune dysregulation and prognostic biomarkers in the context of scrub typhus pathogenesis.

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells.

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation.OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection.METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis.FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae.CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Extracellular vesicles from KSHV-infected endothelial cells activate the complement system.

Extracellular vesicles (EVs), released by cells, are associated with cell-to-cell communication and regulate various cellular processes. EVs draw parallels with viruses for their similar structures and functions. Increasing evidences from recent studies indicate that cells infected with viruses release a variety of EVs. Delineating the functions and mechanisms of EVs released during virus infection is essential for understanding the molecular basis of viral infection and replication as well as associated pathogenesis. The most challenging obstacle for these studies is the separation of EVs from viruses. In this study, we successfully isolated the EVs from de novo Kaposi’s sarcoma-associated herpesvirus (KSHV) infected-human endothelial cells during the period between virus entry and production. Intriguingly, a proteomics analysis of these EVs has revealed alterations of the complement system. Additionally, we have discovered that the EVs from KSHV-infected endothelial cells are potent activators of an alternative pathway of the complement system via exploitation of the endogenous C3 complement protein and properdin. Furthermore, we have found that complement activation promotes KSHV persistent latent infection by activating the NF-κB pathway, which enhances the survival of KSHV-infected cells and inhibits viral lytic replication. Our work identifies a novel role of EVs induced by KSHV during de novo infection and the underlying mechanism of complement activation by EVs.

AXL-Mediated Productive Infection of Human Endothelial Cells by Zika Virus.

The mosquito-borne Zika virus (ZIKV) is now recognized as a blood-borne pathogen, raising an important question about how the virus gets into human bloodstream. The imminent threat of the ZIKV epidemic to the global blood supply also demands novel therapeutics to stop virus transmission though transfusion. We intend to characterize ZIKV tropism for human endothelial cells (ECs) and provide potential targets for intervention. We conducted immunostaining, plaque assay, and quantitative reverse transcription-polymerase chain reaction of ZIKV RNA to evaluate the possible infection of ECs by ZIKV. Both the African and the South American ZIKV strains readily infect human umbilical vein endothelial cells and human ECs derived from aortic and coronary artery, as well as the saphenous vein. Infected ECs released infectious progeny virus. Compared with the African strains, South American ZIKV isolates replicate faster in ECs and are partially cytopathic, suggesting enhanced virulence of these isolates. Flow cytometric analyses showed that the susceptibility of ECs positively correlated with the cell surface levels of tyrosine-protein kinase receptor UFO (AXL) receptor tyrosine kinase. Gain- and loss-of-function studies further revealed that AXL is required for ZIKV entry at a postbinding step. Finally, small-molecule inhibitors of the AXL kinase significantly reduced ZIKA infection of ECs. We identified EC as a key cell type for ZIKV infection. These data support the view of hematogenous dissemination of ZIKV and implicate AXL as a new target for antiviral therapy.

Sequence variations and protein expression levels of the two immune evasion proteins Gpm1 and Pra1 influence virulence of clinical Candida albicans isolates.

Candida albicans, the important human fungal pathogen uses multiple evasion strategies to control, modulate and inhibit host complement and innate immune attack. Clinical C. albicans strains vary in pathogenicity and in serum resistance, in this work we analyzed sequence polymorphisms and variations in the expression levels of two central fungal complement evasion proteins, Gpm1 (phosphoglycerate mutase 1) and Pra1 (pH-regulated antigen 1) in thirteen clinical C. albicans isolates. Four nucleotide (nt) exchanges, all representing synonymous exchanges, were identified within the 747-nt long GPM1 gene. For the 900-nt long PRA1 gene, sixteen nucleotide exchanges were identified, which represented synonymous, as well as non-synonymous exchanges. All thirteen clinical isolates had a homozygous exchange (A to G) at position 73 of the PRA1 gene. Surface levels of Gpm1 varied by 8.2, and Pra1 levels by 3.3 fold in thirteen tested isolates and these differences influenced fungal immune fitness. The high Gpm1/Pra1 expressing candida strains bound the three human immune regulators more efficiently, than the low expression strains. The difference was 44% for Factor H binding, 51% for C4BP binding and 23% for plasminogen binding. This higher Gpm1/Pra1 expressing strains result in enhanced survival upon challenge with complement active, Factor H depleted human serum (difference 40%). In addition adhesion to and infection of human endothelial cells was increased (difference 60%), and C3b surface deposition was less effective (difference 27%). Thus, variable expression levels of central immune evasion protein influences immune fitness of the human fungal pathogen C. albicans and thus contribute to fungal virulence.

Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells.

Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell.

Human cytomegalovirus vaccine based on the envelope gH/gL pentamer complex.

Human Cytomegalovirus (HCMV) utilizes two different pathways for host cell entry. HCMV entry into fibroblasts requires glycoproteins gB and gH/gL, whereas HCMV entry into epithelial and endothelial cells (EC) requires an additional complex composed of gH, gL, UL128, UL130, and UL131A, referred to as the gH/gL-pentamer complex (gH/gL-PC). While there are no established correlates of protection against HCMV, antibodies are thought to be important in controlling infection. Neutralizing antibodies (NAb) that prevent gH/gL-PC mediated entry into EC are candidates to be assessed for in vivo protective function. However, these potent NAb are predominantly directed against conformational epitopes derived from the assembled gH/gL-PC. To address these concerns, we constructed Modified Vaccinia Ankara (MVA) viruses co-expressing all five gH/gL-PC subunits (MVA-gH/gL-PC), subsets of gH/gL-PC subunits (gH/gL or UL128/UL130/UL131A), or the gB subunit from HCMV strain TB40/E. We provide evidence for cell surface expression and assembly of complexes expressing full-length gH or gB, or their secretion when the corresponding transmembrane domains are deleted. Mice or rhesus macaques (RM) were vaccinated three times with MVA recombinants and serum NAb titers that prevented 50% infection of human EC or fibroblasts by HCMV TB40/E were determined. NAb responses induced by MVA-gH/gL-PC blocked HCMV infection of EC with potencies that were two orders of magnitude greater than those induced by MVA expressing gH/gL, UL128-UL131A, or gB. In addition, MVA-gH/gL-PC induced NAb responses that were durable and efficacious to prevent HCMV infection of Hofbauer macrophages, a fetal-derived cell localized within the placenta. NAb were also detectable in saliva of vaccinated RM and reached serum peak levels comparable to NAb titers found in HCMV hyperimmune globulins. This vaccine based on a translational poxvirus platform co-delivers all five HCMV gH/gL-PC subunits to achieve robust humoral responses that neutralize HCMV infection of EC, placental macrophages and fibroblasts, properties of potential value in a prophylactic vaccine.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-γ treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses.

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-β and TNF-α production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-α as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-α and IFN-β as well as the dsRNA analog, poly (I:C). Both IFN-β and TNF-α further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.

Dengue virus infection of SK Hep1 cells: inhibition ofin vitroangiogenesis and altered cytomorphology by expressed viral envelope glycoprotein

Dengue virus (DENV) infection of human endothelial cells has been implicated in the pathobiology of dengue hemorrhagic fever and dengue shock syndrome. However, the mechanisms by which DENV infections alter the functional physiology of endothelial cells remain incompletely understood. In the present study, we examined the susceptibility of a human liver sinusoidal endothelial cell line SK Hep1 to all four serotypes of DENV and studied the effect of the virus on in vitro angiogenesis. All four serotypes of DENV could infect the SK Hep1 cells, but showed variable cytopathic effects, the most pronounced being that of DENV-2. Electron microscopy of the infected cells showed significant ultrastructural changes. In vitro angiogenesis assays on DENV-2 exposed SK Hep1 cells in the matrigel system showed inhibition compared with the controls. Importantly, transfection and transient expression of the DENV-2 envelope glycoprotein (E) in these cells showed drastic alterations in cell shapes and the E protein could be localized by fluorescence microscopy in terminal knob-like structures. Therefore, SK Hep1, a human hepatic sinusoid-derived endothelial cell line, may constitute a potential model to study DENV-endothelial cell interactions in vitro, especially towards understanding the possible virus-induced changes in hepatic endothelium and its role in disease pathogenesis.

The BatR/BatS Two-Component Regulatory System Controls the Adaptive Response ofBartonella henselaeduring Human Endothelial Cell Infection

Here, we report the first comprehensive study of Bartonella henselae gene expression during infection of human endothelial cells. Expression of the main cluster of upregulated genes, comprising the VirB type IV secretion system and its secreted protein substrates, is shown to be under the positive control of the transcriptional regulator BatR. We demonstrate binding of BatR to the promoters of the virB operon and a substrate-encoding gene and provide biochemical evidence that BatR and BatS constitute a functional two-component regulatory system. Moreover, in contrast to the acid-inducible (pH 5.5) homologs ChvG/ChvI of Agrobacterium tumefaciens, BatR/BatS are optimally activated at the physiological pH of blood (pH 7.4). By conservation analysis of the BatR regulon, we show that BatR/BatS are uniquely adapted to upregulate a genus-specific virulence regulon during hemotropic infection in mammals. Thus, we propose that BatR/BatS two-component system homologs represent vertically inherited pH sensors that control the expression of horizontally transmitted gene sets critical for the diverse host-associated life styles of the alphaproteobacteria.

Antiviral effect of Bosentan and Valsartan during coxsackievirus B3 infection of human endothelial cells

In viral myocarditis, adeno- and enteroviruses have most commonly been implicated as causes of infection. Both viruses require the human coxsackie-adenovirus receptor (CAR) to infect the myocardium. Due to its crucial role for viral entry, CAR-downregulation may lead to novel approaches for treatment for viral myocarditis. In this study, we report on pharmaceutical drug influences on CAR levels in human umbilical vein endothelial cells (HUVEC) and cervical carcinoma cells (HeLa) detected by immunoblotting, quantitative real time-PCR and cellular susceptibility to the cardiotropic coxsackie-B3 virus strain Nancy (CVB3). Our results indicate, for the first time, a dose-dependent CAR mRNA and protein downregulation upon Valsartan and Bosentan treatment. Most interestingly, drug-induced CAR diminution significantly reduced the viral load in CVB3-infected HUVEC. In order to assess the regulatory effects of both drugs in detail, we knocked down their protein targets, the G-protein coupled receptors angiotensin-II type-1 receptor (AT(1)R) and endothelin-1 type-A and -B receptors (ET(A)R/ET(B)R) in HUVEC. Receptor-specific gene silencing indicates that CAR gene expression is regulated by agonistic and antagonistic binding to ET(B)R, but not ET(A)R. In addition, neither stimulation nor inhibition of AT(1)R seemed to be involved in CAR gene regulatory processes. Our study indicates that Valsartan and Bosentan protected human endothelial cells from CVB3-infection. Therefore, besides their well-known anti-hypertensive effects these drugs may also protect the myocardium and other tissues from coxsackie- and adenoviral infection.

Essential Role of Mitochondrial Antiviral Signaling, IFN Regulatory Factor (IRF)3, and IRF7 in Chlamydophila pneumoniae-Mediated IFN-β Response and Control of Bacterial Replication in Human Endothelial Cells

Chlamydophila pneumoniae infection of the vascular wall as well as activation of the transcription factor IFN regulatory factor (IRF)3 have been linked to development of chronic vascular lesions and atherosclerosis. The innate immune system detects invading pathogens by use of pattern recognition receptors, some of which are able to stimulate IRF3/7 activation and subsequent type I IFN production (e. g., IFN-beta). In this study, we show that infection of human endothelial cells with C. pneumoniae-induced production of IFN-beta, a cytokine that so far has been mainly associated with antiviral immunity. Moreover, C. pneumoniae infection led to IRF3 and IRF7 nuclear translocation in HUVECs and RNA interference experiments showed that IRF3 and IRF7 as well as the mitochondrial antiviral signaling (MAVS) were essential for IFN-beta induction. Finally, C. pneumoniae replication was enhanced in endothelial cells in which IRF3, IRF7, or MAVS expression was inhibited by small interfering RNA and attenuated by IFN-beta treatment. In conclusion, C. pneumoniae infection of endothelial cells activates an MAVS-, IRF3-, and IRF7-dependent signaling, which controls bacterial growth and might modulate development of vascular lesions.

Robust inference of the context specific structure and temporal dynamics of gene regulatory network

BackgroundResponse of cells to changing endogenous or exogenous conditions is governed by intricate molecular interactions, or regulatory networks. To lead to appropriate responses, regulatory network should be 1) context-specific, i.e., its constituents and topology depend on the phonotypical and experimental context including tissue types and cell conditions, such as damage, stress, macroenvironments of cell, etc. and 2) time varying, i.e., network elements and their regulatory roles change actively over time to control the endogenous cell states e.g. different stages in a cell cycle.ResultsA novel network model PathRNet and a reconstruction approach PATTERN are proposed for reconstructing the context specific time varying regulatory networks by integrating microarray gene expression profiles and existing knowledge of pathways and transcription factors. The nodes of the PathRNet are Transcription Factors (TFs) and pathways, and edges represent the regulation between pathways and TFs. The reconstructed PathRNet for Kaposi's sarcoma-associated herpesvirus infection of human endothelial cells reveals the complicated dynamics of the underlying regulatory mechanisms that govern this intricate process. All the related materials including source code are available at http://compgenomics.utsa.edu/tvnet.html.ConclusionsThe proposed PathRNet provides a system level landscape of the dynamics of gene regulatory circuitry. The inference approach PATTERN enables robust reconstruction of the temporal dynamics of pathway-centric regulatory networks. The proposed approach for the first time provides a dynamic perspective of pathway, TF regulations, and their interaction related to specific endogenous and exogenous conditions.

Andes Virus Recognition of Human and Syrian Hamster β 3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human alpha(v)beta(3) integrins are receptors for several pathogenic hantaviruses, and the function of alpha(v)beta(3) integrins on endothelial cells suggests a role for alpha(v)beta(3) in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity alpha(v)beta(3) integrin ligand vitronectin and by antibodies to alpha(v)beta(3) integrins. Further, antibodies to the beta(3) integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster beta(3) subunits, but not murine or bovine beta(3), inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with beta(3) subunits through PSI domain residues conserved in both Syrian hamster and human beta(3) integrins. Sequencing the Syrian hamster beta(3) integrin PSI domain revealed eight differences between Syrian hamster and human beta(3) integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine beta(3) integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human beta(3) PSI domain to contain the L33P substitution present in bovine beta(3) integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human beta(3) permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine beta(3) integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster alpha(v)beta(3) integrins are key receptors for ANDV and that specific residues within the beta(3) integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human beta(3) polymorphism, these findings further suggest the importance of specific beta(3) integrin residues in hantavirus infection. These findings rationalize determining the role of beta(3) integrins in hantavirus pathogenesis in the Syrian hamster model.

Pichinde virus induces microvascular endothelial cell permeability through the production of nitric oxide

This report is the first to demonstrate infection of human endothelial cells by Pichinde virus (PIC). PIC infection induces an upregulation of the inducible nitric oxide synthase gene; as well as an increase in detectable nitric oxide (NO). PIC induces an increase in permeability in endothelial cell monolayers which can be abrogated at all measured timepoints with the addition of a nitric oxide synthase inhibitor, indicating a role for NO in the alteration of endothelial barrier function. Because NO has shown antiviral activity against some viruses, viral titer was measured after addition of the NO synthase inhibitor and found to have no effect in altering virus load in infected EC. The NO synthase inhibition also has no effect on levels of activated caspases induced by PIC infection. Taken together, these data indicate NO production induced by Pichinde virus infection has a pathogenic effect on endothelial cell monolayer permeability.