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1.  Hypoxia Induces Permeability and Giant Cell Responses of Andes Virus-Infected Pulmonary Endothelial Cells by Activating the mTOR-S6K Signaling Pathway 
Journal of Virology  2013;87(23):12999-13008.
Andes virus (ANDV) is a South American hantavirus that causes a highly lethal hantavirus pulmonary syndrome (HPS) characterized by hypoxia, thrombocytopenia, and vascular leakage leading to acute pulmonary edema. ANDV infects human pulmonary microvascular and lymphatic endothelial cells (MECs and LECs, respectively) and nonlytically enhances the permeability of interendothelial cell adherence junctions in response to vascular endothelial growth factor (VEGF). Recent findings also indicate that ANDV causes the formation of giant endothelial cells. Here, we demonstrate that hypoxic conditions alone enhance permeability and giant cell responses of ANDV-infected MECs and LECs through activation of the mTOR signaling pathway. In contrast to infection of cells with nonpathogenic Tula virus (TULV), we observed that exposure of ANDV-infected MECs and LECs to hypoxic conditions resulted in a 3- to 6-fold increase in monolayer permeability and the formation of giant cells 3× to 5× normal size. ANDV infection in combination with hypoxic conditions resulted in the enhancement of hypoxia-inducible factor 1α (HIF1α)-directed VEGF A, angiopoietin 4, and EGLN3 transcriptional responses. Constitutive mTOR signaling induces the formation of giant cells via phosphorylation of S6K, and mTOR regulates hypoxia and VEGF A-induced cellular responses. We found that S6K was hyperphosphorylated in ANDV-infected, hypoxia-treated MECs and LECs and that rapamycin treatment for 1 h inhibited mTOR signaling responses and blocked permeability and giant cell formation in ANDV-infected monolayers. These findings indicate that ANDV infection and hypoxic conditions enhance mTOR signaling responses, resulting in enhanced endothelial cell permeability and suggest a role for rapamycin in therapeutically stabilizing the endothelium of microvascular and lymphatic vessels during ANDV infection.
doi:10.1128/JVI.02103-13
PMCID: PMC3838155  PMID: 24067973
2.  Andes Virus Infection of Lymphatic Endothelial Cells Causes Giant Cell and Enhanced Permeability Responses That Are Rapamycin and Vascular Endothelial Growth Factor C Sensitive 
Journal of Virology  2012;86(16):8765-8772.
Hantaviruses primarily infect endothelial cells (ECs) and nonlytically cause vascular changes that result in hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Acute pulmonary edema during HPS may be caused by capillary leakage and failure of lymphatic vessels to clear fluids. Uniquely regulated lymphatic ECs (LECs) control fluid clearance, although roles for lymphatics in hantavirus disease remain undetermined. Here we report that hantaviruses productively infect LECs and that LEC infection by HPS causing Andes virus (ANDV) and HFRS causing Hantaan virus (HTNV) are inhibited by αvβ3 integrin antibodies. Although αvβ3 integrins regulate permeabilizing responses directed by vascular endothelial growth factor receptor 2 (VEGFR2), we found that only ANDV-infected LECs were hyperpermeabilized by the addition of VEGF-A. However, VEGF-C activation of LEC-specific VEGFR3 receptors blocked ANDV- and VEGF-A-induced LEC permeability. In addition, ∼75% of ANDV-infected LECs became viable mononuclear giant cells, >4 times larger than normal, in response to VEGF-A. Giant cells are associated with constitutive mammalian target of rapamycin (mTOR) activation, and we found that both giant LECs and LEC permeability were sensitive to rapamycin, an mTOR inhibitor, and VEGF-C addition. These findings indicate that ANDV uniquely alters VEGFR2-mTOR signaling responses of LECs, resulting in giant cell and LEC permeability responses. This suggests that ANDV infection alters normal LEC and lymphatic vessel functions which may contribute to edematous fluid accumulation during HPS. Moreover, the ability of VEGF-C and rapamycin to normalize LEC responses suggests a potential therapeutic approach for reducing pulmonary edema and the severity of HPS following ANDV infection.
doi:10.1128/JVI.00817-12
PMCID: PMC3421700  PMID: 22696643
3.  Elevated VEGF Levels in Pulmonary Edema Fluid and PBMCs from Patients with Acute Hantavirus Pulmonary Syndrome 
Advances in Virology  2012;2012:674360.
Hantavirus pulmonary syndrome is characterized by vascular permeability, hypoxia, and acute pulmonary edema. Vascular endothelial growth factor (VEGF) is induced by hypoxia, potently induces vascular permeability, and is associated with high-altitude-induced pulmonary edema. Hantaviruses alter the normal regulation of β3 integrins that restrict VEGF-directed permeability and hantavirus infected endothelial cells are hyperresponsive to the permeabilizing effects of VEGF. However, the role of VEGF in acute pulmonary edema observed in HPS patients remains unclear. Here we retrospectively evaluate VEGF levels in pulmonary edema fluid (PEF), plasma, sera, and PBMCs from 31 HPS patients. VEGF was elevated in HPS patients PEF compared to controls with the highest levels observed in PEF samples from a fatal HPS case. VEGF levels were highest in PBMC samples during the first five days of hospitalization and diminished during recovery. Significantly increased PEF and PBMC VEGF levels are consistent with acute pulmonary edema observed in HPS patients and HPS disease severity. We observed substantially lower VEGF levels in a severe HPS disease survivor after extracorporeal membrane oxygenation. These findings suggest the importance of patients' VEGF levels during HPS, support the involvement of VEGF responses in HPS pathogenesis, and suggest targeting VEGF responses as a potential therapeutic approach.
doi:10.1155/2012/674360
PMCID: PMC3432326  PMID: 22956954
4.  The Role of the Endothelium in HPS Pathogenesis and Potential Therapeutic Approaches 
Advances in Virology  2012;2012:467059.
American hantaviruses cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect endothelial cells and cause dramatic changes in barrier functions of the endothelium without disrupting the endothelium. Instead hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions of capillaries. The endothelium of arteries, veins, and lymphatic vessels is unique and central to the function of vast pulmonary capillary beds, which regulate pulmonary fluid accumulation. The endothelium maintains vascular barrier functions through a complex series of redundant receptors and signaling pathways that serve to both permit fluid and immune cell efflux into tissues and restrict tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to alter capillary permeability but also defines potential therapeutic targets for regulating acute pulmonary edema and HPS disease. Here we discuss interactions of HPS causing hantaviruses with the endothelium, potential endothelial cell-directed permeability mechanisms, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease.
doi:10.1155/2012/467059
PMCID: PMC3395186  PMID: 22811711
5.  The C-Terminal 42 Residues of the Tula Virus Gn Protein Regulate Interferon Induction▿ 
Journal of Virology  2011;85(10):4752-4760.
Hantaviruses primarily infect the endothelial cell lining of capillaries and cause two vascular permeability-based diseases. The ability of pathogenic hantaviruses to regulate the early induction of interferon determines whether hantaviruses replicate in endothelial cells. Tula virus (TULV) and Prospect Hill virus (PHV) are hantaviruses which infect human endothelial cells but fail to cause human disease. PHV is unable to inhibit early interferon (IFN) responses and fails to replicate within human endothelial cells. However, TULV replicates successfully in human endothelial cells, suggesting that TULV is capable of regulating cellular IFN responses. We observed a >300-fold reduction in the IFN-stimulated genes (ISGs) MxA and ISG56 following TULV versus PHV infection of endothelial cells 1 day postinfection. Similar to results with pathogenic hantaviruses, expressing the TULV Gn protein cytoplasmic tail (Gn-T) blocked RIG-I- and TBK1-directed transcription from IFN-stimulated response elements (ISREs) and IFN-β promoters (>90%) but not transcription directed by constitutively active IFN regulatory factor-3 (IRF3). In contrast, expressing the PHV Gn-T had no effect on TBK1-induced transcriptional responses. Analysis of Gn-T truncations demonstrated that the C-terminal 42 residues of the Gn-T (Gn-T-C42) from TULV, but not PHV, inhibited IFN induction >70%. These findings demonstrate that the TULV Gn-T inhibits IFN- and ISRE-directed responses upstream of IRF3 at the level of the TBK1 complex and further define a 42-residue domain of the TULV Gn-T that inhibits IFN induction. In contrast to pathogenic hantavirus Gn-Ts, the TULV Gn-T lacks a C-terminal degron domain and failed to bind tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), a TBK1 complex component required for IRF3 activation. These findings indicate that the nonpathogenic TULV Gn-T regulates IFN induction but accomplishes this via unique interactions with cellular TBK1 complexes. These findings fundamentally distinguish nonpathogenic hantaviruses, PHV and TULV, and demonstrate that IFN regulation alone is insufficient for hantaviruses to cause disease. Yet regulating the early IFN response is necessary for hantaviruses to replicate within human endothelial cells and to be pathogenic. Thus, in addition to IFN regulation, hantaviruses contain discrete virulence determinants which permit them to be human pathogens.
doi:10.1128/JVI.01945-10
PMCID: PMC3126157  PMID: 21367904
6.  VEGFR2 and Src Kinase Inhibitors Suppress Andes Virus-Induced Endothelial Cell Permeability ▿  
Journal of Virology  2010;85(5):2296-2303.
Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ∼70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC50s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.
doi:10.1128/JVI.02319-10
PMCID: PMC3067787  PMID: 21177802
7.  Andes Virus Regulation of Cellular MicroRNAs Contributes to Hantavirus-Induced Endothelial Cell Permeability▿  
Journal of Virology  2010;84(22):11929-11936.
Hantaviruses infect human endothelial cells (ECs) and cause two diseases marked by vascular permeability defects, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Vascular permeability occurs in the absence of EC lysis, suggesting that hantaviruses alter normal EC fluid barrier functions. ECs infected by pathogenic hantaviruses are hyperresponsive to vascular endothelial growth factor (VEGF), and this alters the fluid barrier function of EC adherens junctions, resulting in enhanced paracellular permeability. Vascular permeability and VEGF-directed responses are determined by EC-specific microRNAs (miRNAs), which regulate cellular mRNA transcriptional responses. miRNAs mature within cytoplasmic processing bodies (P bodies), and the hantavirus nucleocapsid (N) protein binds RNA and localizes to P bodies, suggesting that hantaviruses may modify miRNA functions within infected ECs. Here we assessed changes in EC miRNAs following infection by the HPS-causing Andes hantavirus (ANDV). We analyzed 352 human miRNAs within ANDV-infected ECs using quantitative real-time (RT)-PCR arrays. Fourteen miRNAs, including six miRNAs that are associated with regulating vascular integrity, were upregulated >4-fold following infection by ANDV. Nine miRNAs were downregulated 3- to 3,400-fold following ANDV infection; these included miR-410, involved in regulating secretion, and miR-218, which is linked to the regulation of EC migration and vascular permeability. We further analyzed changes in miR-126, an EC-specific miRNA that regulates vascular integrity by suppressing SPRED1 and PIK3R2 mRNAs. While miR-126 levels were only slightly altered, we found that SPRED1 and PIK3R2 mRNA levels were increased 10- and 7-fold, respectively, in ANDV-infected ECs but were unaltered in ECs infected by the nonpathogenic Tula hantavirus (TULV). Consistent with increased SPRED1 expression, we found that the level of phospho-cofilin was decreased within ANDV-infected ECs. Moreover, small interfering RNA (siRNA) knockdown of SPRED1 dramatically decreased the permeability of ANDV-infected ECs in response to VEGF, suggesting that increased SPRED1 contributes to EC permeability following ANDV infection. These findings suggest that interference with normal miRNA functions contributes to the enhanced paracellular permeability of ANDV-infected ECs and that hantavirus regulation of miRNA functions is an additional determinant of hantavirus pathogenesis.
doi:10.1128/JVI.01658-10
PMCID: PMC2977893  PMID: 20844033
8.  Pathogenic Hantaviruses Andes Virus and Hantaan Virus Induce Adherens Junction Disassembly by Directing Vascular Endothelial Cadherin Internalization in Human Endothelial Cells▿  
Journal of Virology  2010;84(14):7405-7411.
Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.
doi:10.1128/JVI.00576-10
PMCID: PMC2898267  PMID: 20463083
9.  Pathogenic Hantaviruses Direct the Adherence of Quiescent Platelets to Infected Endothelial Cells▿  
Journal of Virology  2010;84(9):4832-4839.
Hantavirus infections are noted for their ability to infect endothelial cells, cause acute thrombocytopenia, and trigger 2 vascular-permeability-based diseases. However, hantavirus infections are not lytic, and the mechanisms by which hantaviruses cause capillary permeability and thrombocytopenia are only partially understood. The role of β3 integrins in hemostasis and the inactivation of β3 integrin receptors by pathogenic hantaviruses suggest the involvement of hantaviruses in altered platelet and endothelial cell functions that regulate permeability. Here, we determined that pathogenic hantaviruses bind to quiescent platelets via a β3 integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to β3 integrin receptors prevents platelet activation. The ability of hantaviruses to bind platelets further suggested that cell-associated hantaviruses might recruit platelets to the endothelial cell surface. Our findings indicate that Andes virus (ANDV)- or Hantaan virus (HTNV)-infected endothelial cells specifically direct the adherence of calcein-labeled platelets. In contrast, cells comparably infected with nonpathogenic Tula virus (TULV) failed to recruit platelets to the endothelial cell surface. Platelet adherence was dependent on endothelial cell β3 integrins and neutralized by the addition of the anti-β3 Fab fragment, c7E3, or specific ANDV- or HTNV-neutralizing antibodies. These findings indicate that pathogenic hantaviruses displayed on the surface of infected endothelial cells bind platelets and that a platelet layer covers the surface of infected endothelial cells. This fundamentally changes the appearance of endothelial cells and has the potential to alter cellular immune responses, platelet activation, and endothelial cell functions that affect vascular permeability. Hantavirus-directed platelet quiescence and recruitment to vast endothelial cell beds further suggests mechanisms by which hantaviruses may cause thrombocytopenia and induce hypoxia. These findings are fundamental to our understanding of pathogenic-hantavirus regulation of endothelial cell responses that contribute to vascular permeability.
doi:10.1128/JVI.02405-09
PMCID: PMC2863738  PMID: 20181715
10.  Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain ▿  
Journal of Virology  2009;84(1):352-360.
Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human αvβ3 integrins are receptors for several pathogenic hantaviruses, and the function of αvβ3 integrins on endothelial cells suggests a role for αvβ3 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 αvβ3 integrin ligand vitronectin and by antibodies to αvβ3 integrins. Further, antibodies to the β3 integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β3 subunits, but not murine or bovine β3, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β3 subunits through PSI domain residues conserved in both Syrian hamster and human β3 integrins. Sequencing the Syrian hamster β3 integrin PSI domain revealed eight differences between Syrian hamster and human β3 integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β3 integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β3 PSI domain to contain the L33P substitution present in bovine β3 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 β3 permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β3 integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster αvβ3 integrins are key receptors for ANDV and that specific residues within the β3 integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β3 polymorphism, these findings further suggest the importance of specific β3 integrin residues in hantavirus infection. These findings rationalize determining the role of β3 integrins in hantavirus pathogenesis in the Syrian hamster model.
doi:10.1128/JVI.01013-09
PMCID: PMC2798441  PMID: 19846530
11.  The NY-1 Hantavirus Gn Cytoplasmic Tail Coprecipitates TRAF3 and Inhibits Cellular Interferon Responses by Disrupting TBK1-TRAF3 Complex Formation▿  
Journal of Virology  2008;82(18):9115-9122.
Pathogenic hantaviruses replicate within human endothelial cells and cause two diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. In order to replicate in endothelial cells pathogenic hantaviruses inhibit the early induction of beta interferon (IFN-β). Expression of the cytoplasmic tail of the pathogenic NY-1 hantavirus Gn protein is sufficient to inhibit RIG-I- and TBK1-directed IFN responses. The formation of TBK1-TRAF3 complexes directs IRF-3 phosphorylation, and both IRF-3 and NF-κB activation are required for transcription from the IFN-β promoter. Here we report that the NY-1 virus (NY-1V) Gn tail inhibits both TBK1-directed NF-κB activation and TBK1-directed transcription from promoters containing IFN-stimulated response elements. The NY-1V Gn tail coprecipitated TRAF3 from cellular lysates, and analysis of TRAF3 deletion mutants demonstrated that the TRAF3 N terminus is sufficient for interacting with the NY-1V Gn tail. In contrast, the Gn tail of the nonpathogenic hantavirus Prospect Hill virus (PHV) failed to coprecipitate TRAF3 or inhibit NF-κB or IFN-β transcriptional responses. Further, expression of the NY-1V Gn tail blocked TBK1 coprecipitation of TRAF3 and infection by NY-1V, but not PHV, blocked the formation of TBK1-TRAF3 complexes. These findings indicate that the NY-1V Gn cytoplasmic tail forms a complex with TRAF3 which disrupts the formation of TBK1-TRAF3 complexes and downstream signaling responses required for IFN-β transcription.
doi:10.1128/JVI.00290-08
PMCID: PMC2546897  PMID: 18614628
12.  Hantaviruses Direct Endothelial Cell Permeability by Sensitizing Cells to the Vascular Permeability Factor VEGF, while Angiopoietin 1 and Sphingosine 1-Phosphate Inhibit Hantavirus-Directed Permeability▿  
Journal of Virology  2008;82(12):5797-5806.
Hantaviruses infect human endothelial cells and cause two vascular permeability-based diseases: hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Hantavirus infection alone does not permeabilize endothelial cell monolayers. However, pathogenic hantaviruses inhibit the function of αvβ3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional β3 integrins that normally regulate permeabilizing vascular endothelial growth factor (VEGF) responses. Here we show that pathogenic Hantaan, Andes, and New York-1 hantaviruses dramatically enhance the permeability of endothelial cells in response to VEGF, while the nonpathogenic hantaviruses Prospect Hill and Tula have no effect on endothelial cell permeability. Pathogenic hantaviruses directed endothelial cell permeability 2 to 3 days postinfection, coincident with pathogenic hantavirus inhibition of αvβ3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to αvβ3 integrin-deficient cells, specifically enhance VEGF-directed permeabilizing responses. Using the hantavirus permeability assay we further demonstrate that the endothelial-cell-specific growth factor angiopoietin 1 (Ang-1) and the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) inhibit hantavirus directed endothelial cell permeability at physiologic concentrations. These results demonstrate the utility of a hantavirus permeability assay and rationalize the testing of Ang-1, S1P, and antibodies to VEGFR2 as potential hantavirus therapeutics. The central importance of β3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering β3 or VEGF responses may be a common feature of additional viral hemorrhagic diseases. As a result, our findings provide a potential mechanism for vascular leakage after infection by pathogenic hantaviruses and the means to inhibit hantavirus-directed endothelial cell permeability that may be applicable to additional vascular leak syndromes.
doi:10.1128/JVI.02397-07
PMCID: PMC2395149  PMID: 18367532
14.  The Pathogenic NY-1 Hantavirus G1 Cytoplasmic Tail Inhibits RIG-I- and TBK-1-Directed Interferon Responses 
Journal of Virology  2006;80(19):9676-9686.
Hantaviruses cause two diseases with prominent vascular permeability defects, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. All hantaviruses infect human endothelial cells, although it is unclear what differentiates pathogenic from nonpathogenic hantaviruses. We observed dramatic differences in interferon-specific transcriptional responses between pathogenic and nonpathogenic hantaviruses at 1 day postinfection, suggesting that hantavirus pathogenesis may in part be determined by viral regulation of cellular interferon responses. In contrast to pathogenic NY-1 virus (NY-1V) and Hantaan virus (HTNV), nonpathogenic Prospect Hill virus (PHV) elicits early interferon responses following infection of human endothelial cells. We determined that PHV replication is blocked in human endothelial cells and that RNA and protein synthesis by PHV, but not NY-1V or HTNV, is inhibited at 2 to 4 days postinfection. The addition of antibodies to beta interferon (IFN-β) blocked interferon-directed MxA induction by >90% and demonstrated that hantavirus infection induces the secretion of IFN-β from endothelial cells. Coinfecting endothelial cells with NY-1V and PHV resulted in a 60% decrease in the induction of interferon-responsive MxA transcripts by PHV and further suggested the potential for NY-1V to regulate early IFN responses. Expression of the NY-1V G1 cytoplasmic tail inhibited by >90% RIG-I- and downstream TBK-1-directed transcription from interferon-stimulated response elements or β-interferon promoters in a dose-dependent manner. In contrast, expression of the NY-1V nucleocapsid or PHV G1 tail had no effect on RIG-I- or TBK-1-directed transcriptional responses. Further, neither the NY-1V nor PHV G1 tails inhibited transcriptional responses directed by a constitutively active form of interferon regulatory factor 3 (IRF-3 5D), and IRF-3 is a direct target of TBK-1 phosphorylation. These findings indicate that the pathogenic NY-1V G1 protein regulates cellular IFN responses upstream of IRF-3 phosphorylation at the level of the TBK-1 complex. These findings further suggest that the G1 cytoplasmic tail contains a virulence element which determines the ability of hantaviruses to bypass innate cellular immune responses and delineates a mechanism for pathogenic hantaviruses to successfully replicate within human endothelial cells.
doi:10.1128/JVI.00508-06
PMCID: PMC1617216  PMID: 16973572
15.  Discrete Domains within the Rotavirus VP5* Direct Peripheral Membrane Association and Membrane Permeability 
Journal of Virology  2004;78(4):2037-2044.
Cleavage of the rotavirus spike protein, VP4, is required for rotavirus-induced membrane permeability and viral entry into cells. The VP5* cleavage product selectively permeabilizes membranes and liposomes and contains an internal hydrophobic domain that is required for membrane permeability. Here we investigate VP5* domains (residues 248 to 474) that direct membrane binding. We determined that expressed VP5 fragments containing residues 248 to 474 or 265 to 474, including the internal hydrophobic domain, bind to cellular membranes but are not present in Triton X-100-resistant membrane rafts. Expressed VP5 partitions into aqueous but not detergent phases of Triton X-114, suggesting that VP5 is not integrally inserted into membranes. Since high-salt or alkaline conditions eluted VP5 from membranes, our findings demonstrate that VP5 is peripherally associated with membranes. Interestingly, mutagenesis of residue 394 (W→R) within the VP5 hydrophobic domain, which abolishes VP5-directed permeability, had no effect on VP5's peripheral membrane association. In contrast, deletion of N-terminal VP5 residues (residues 265 to 279) abolished VP5 binding to membranes. Alanine mutagenesis of two positively charged residues within this domain (residues 274R and 276K) dramatically reduced (>95%) binding of VP5 to membranes and suggested their potential interaction with polar head groups of the lipid bilayer. Mutations in either the VP5 hydrophobic or basic domain blocked VP5-directed permeability of cells. These findings indicate that there are at least two discrete domains within VP5* required for pore formation: an N-terminal basic domain that permits VP5* to peripherally associate with membranes and an internal hydrophobic domain that is essential for altering membrane permeability. These results provide a fundamental understanding of interactions between VP5* and the membrane, which are required for rotavirus entry.
doi:10.1128/JVI.78.4.2037-2044.2004
PMCID: PMC369428  PMID: 14747568

Results 1-15 (15)