Hypoxia induces permeability and giant cell responses of Andes virus-infected pulmonary endothelial cells by activating the mTOR-S6K signaling pathway.

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.

Andes virus infection of lymphatic endothelial cells causes giant cell and enhanced permeability responses that are rapamycin and vascular endothelial growth factor C sensitive.

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 α(v)ß(3) integrin antibodies. Although α(v)ß(3) 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.

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability.

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 (IC(50)s), 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.

The C-terminal 42 residues of the Tula virus Gn protein regulate interferon induction.

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-ß 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.

Pathogenic hantaviruses Andes virus and Hantaan virus induce adherens junction disassembly by directing vascular endothelial cadherin internalization in human endothelial cells.

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.

Pathogenic hantaviruses direct the adherence of quiescent platelets to infected endothelial cells.

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 beta(3) integrins in hemostasis and the inactivation of beta(3) 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 beta(3) integrin-dependent mechanism. This suggests that platelets may contribute to hantavirus dissemination within infected patients and provides a means by which hantavirus binding to beta(3) 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 beta(3) integrins and neutralized by the addition of the anti-beta(3) 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.

Andes virus regulation of cellular microRNAs contributes to hantavirus-induced endothelial cell permeability.

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.

Andes virus recognition of human and Syrian hamster beta3 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.

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.

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 alphav beta3 integrins on endothelial cells, and hemorrhagic disease and vascular permeability deficits are consequences of dysfunctional beta3 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 alphav beta3 integrin functions, and hantavirus-directed permeability was inhibited by antibodies to VEGF receptor 2 (VEGFR2). These studies demonstrate that pathogenic hantaviruses, similar to alphav beta3 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 beta3 integrins and VEGF responses in vascular leak and hemorrhagic disease further suggest that altering beta3 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.

The NY-1 hantavirus Gn cytoplasmic tail coprecipitates TRAF3 and inhibits cellular interferon responses by disrupting TBK1-TRAF3 complex formation.

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-beta). 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-kappaB activation are required for transcription from the IFN-beta promoter. Here we report that the NY-1 virus (NY-1V) Gn tail inhibits both TBK1-directed NF-kappaB 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-kappaB or IFN-beta 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-beta transcription.

The Andes Virus Nucleocapsid Protein Directs Basal Endothelial Cell Permeability by Activating RhoA.

Andes virus (ANDV) predominantly infects microvascular endothelial cells (MECs) and nonlytically causes an acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients, virtually every pulmonary MEC is infected, MECs are enlarged, and infection results in vascular leakage and highly lethal pulmonary edema. We observed that MECs infected with the ANDV hantavirus or expressing the ANDV nucleocapsid (N) protein showed increased size and permeability by activating the Rheb and RhoA GTPases. Expression of ANDV N in MECs increased cell size by preventing tuberous sclerosis complex (TSC) repression of Rheb-mTOR-pS6K. N selectively bound the TSC2 N terminus (1 to 1403) within a complex containing TSC2/TSC1/TBC1D7, and endogenous TSC2 reciprocally coprecipitated N protein from ANDV-infected MECs. TSCs normally restrict RhoA-induced MEC permeability, and we found that ANDV infection or N protein expression constitutively activated RhoA. This suggests that the ANDV N protein alone is sufficient to activate signaling pathways that control MEC size and permeability. Further, RhoA small interfering RNA, dominant-negative RhoA(N19), and the RhoA/Rho kinase inhibitors fasudil and Y27632 dramatically reduced the permeability of ANDV-infected MECs by 80 to 90%. Fasudil also reduced the bradykinin-directed permeability of ANDV and Hantaan virus-infected MECs to control levels. These findings demonstrate that ANDV activation of RhoA causes MEC permeability and reveal a potential edemagenic mechanism for ANDV to constitutively inhibit the basal barrier integrity of infected MECs. The central importance of RhoA activation in MEC permeability further suggests therapeutically targeting RhoA, TSCs, and Rac1 as potential means of resolving capillary leakage during hantavirus infections. IMPORTANCE: HPS is hallmarked by acute pulmonary edema, hypoxia, respiratory distress, and the ubiquitous infection of pulmonary MECs that occurs without disrupting the endothelium. Mechanisms of MEC permeability and targets for resolving lethal pulmonary edema during HPS remain enigmatic. Our findings suggest a novel underlying mechanism of MEC dysfunction resulting from ANDV activation of the Rheb and RhoA GTPases that, respectively, control MEC size and permeability. Our studies show that inhibition of RhoA blocks ANDV-directed permeability and implicate RhoA as a potential therapeutic target for restoring capillary barrier function to the ANDV-infected endothelium. Since RhoA activation forms a downstream nexus for factors that cause capillary leakage, blocking RhoA activation is liable to restore basal capillary integrity and prevent edema amplified by tissue hypoxia and respiratory distress. Targeting the endothelium has the potential to resolve disease during symptomatic stages, when replication inhibitors lack efficacy, and to be broadly applicable to other hemorrhagic and edematous viral diseases.

Endothelial cell dysfunction in viral hemorrhage and edema.

The endothelium maintains a vascular barrier by controlling platelet and immune cell interactions, capillary tone and interendothelial cell (EC) adherence. Here we suggest common elements in play during viral infection of the endothelium that alter normal EC functions and contribute to lethal hemorrhagic or edematous diseases. In viral reservoir hosts, infection of capillaries and lymphatic vessels may direct immunotolerance without disease, but in the absence of these cognate interactions they direct the delayed onset of human disease characterized by thrombocytopenia and vascular leakage in a severe endothelial dysfunction syndrome. Here we present insight into EC controls of hemostasis, immune response and capillary permeability that are altered by viral infection of the endothelium.

Slit2-Robo4 receptor responses inhibit ANDV directed permeability of human lung microvascular endothelial cells.

Hantaviruses nonlytically infect human endothelial cells (ECs) and cause edematous and hemorrhagic diseases. Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS), and Hantaan virus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses enhance vascular endothelial growth factor directed EC permeability resulting in the disassembly of inter-endothelial cell adherens junctions (AJs). Recent studies demonstrate that Slit2 binding to Robo1/Robo4 receptors on ECs has opposing effects on AJ disassembly and vascular fluid barrier functions. Here we demonstrate that Slit2 inhibits ANDV and HTNV induced permeability and AJ disassembly of pulmonary microvascular ECs (PMECs) by interactions with Robo4. In contrast, Slit2 had no effect on the permeability of ANDV infected human umbilical vein ECs (HUVECs). Analysis of Robo1/Robo4 expression determined that PMECs express Robo4, but not Robo1, while HUVECs expressed both Robo4 and Robo1 receptors. SiRNA knockdown of Robo4 in PMECs prevented Slit2 inhibition of ANDV induced permeability demonstrating that Robo4 receptors determine PMEC responsiveness to Slit2. Collectively, this data demonstrates a selective role for Slit2/Robo4 responses within PMECs that inhibits ANDV induced permeability and AJ disassembly. These findings suggest Slit2s utility as a potential HPS therapeutic that stabilizes the pulmonary endothelium and antagonizes ANDV induced pulmonary edema.

Role of vascular and lymphatic endothelial cells in hantavirus pulmonary syndrome suggests targeted therapeutic approaches.

BACKGROUND: Hantaviruses in the Americas cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect microvascular and lymphatic endothelial cells and cause dramatic changes in barrier functions without disrupting the endothelium. Hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions. The endothelium of arteries, veins, and lymphatic vessels are unique and central to the function of vast pulmonary capillary beds that regulate pulmonary fluid accumulation. RESULTS: We have found that HPS-causing hantaviruses alter vascular barrier functions of microvascular and lymphatic endothelial cells by altering receptor and signaling pathway responses that serve to permit fluid tissue influx and clear tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to cause acute pulmonary edema, as well as potential therapeutic targets for reducing the severity of HPS disease. CONCLUSIONS: Here we discuss interactions of HPS-causing hantaviruses with the endothelium, roles for unique lymphatic endothelial responses in HPS, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease.

Hantavirus regulation of endothelial cell functions.

Hantaviruses cause two vascular permeability-based diseases and primarily infect endothelial cells which form the primary fluid barrier of the vasculature. Since hantavirus infections are not lytic, the mechanisms by which hantaviruses cause haemorrhagic fever with renal syndrome (HFRS) or Hantavirus Pulmonary Syndrome (HPS) are indeterminate. HPS is associated with acute pulmonary oedema and HFRS with moderate haemorrhage and renal sequelae, perhaps reflecting the location of vast microvascular beds and endothelial cell reservoirs available for hantavirus infection. Endothelial cells regulate capillary integrity, and hantavirus infection provides a primary means for altering vascular permeability that contributes to pathogenesis. The central importance of endothelial cells in regulating oedema, vascular repair, angiogenesis, immune cell recruitment, platelet deposition as well as gas exchange and solute delivery suggest that a multitude of inputs and cellular responses may be influenced by hantavirus infection and contribute to pathogenic changes in vascular permeability. Here we focus on understanding hantavirus interactions with endothelial cells which are linked to vascular permeability, and provide insight into the contribution of endothelial cell responses in hantavirus pathogenesis.

The pathogenic NY-1 hantavirus G1 cytoplasmic tail inhibits RIG-I- and TBK-1-directed interferon responses.

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-beta) blocked interferon-directed MxA induction by >90% and demonstrated that hantavirus infection induces the secretion of IFN-beta 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 beta-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.

Pathogenic hantaviruses bind plexin-semaphorin-integrin domains present at the apex of inactive, bent alphavbeta3 integrin conformers.

The alphavbeta3 integrins are linked to human bleeding disorders, and pathogenic hantaviruses regulate the function of alphavbeta3 integrins and cause acute vascular diseases. alphavbeta3 integrins are present in either extended (active) or dramatically bent (inactive) structures, and interconversion of alphavbeta3 conformers dynamically regulates integrin functions. Here, we show that hantaviruses bind human alphavbeta3 integrins and that binding maps to the plexin-semaphorin-integrin (PSI) domain present at the apex of inactive, bent, alphavbeta3-integrin structures. Pathogenic hantaviruses [New York-1 virus (NY-1V) and Hantaan virus (HTNV)] bind immobilized beta3 polypeptides containing the PSI domain, and human (but not murine) beta3 polypeptides inhibit hantavirus infectivity. Substitution of human beta3 residues 1-39 for murine beta3 residues directed pathogenic hantavirus infection of nonpermissive CHO cells expressing chimeric alphavbeta3 receptors. Mutation of murine beta3 Asn-39 to Asp-39 present in human beta3 homologues (N39D) permitted hantavirus infection of cells and specified PSI domain residue interactions with pathogenic hantaviruses. In addition, cell-surface expression of alphavbeta3 locked in an inactive bent conformation conferred hantavirus infectivity of CHO cells. Our findings indicate that hantaviruses bind to a unique domain exposed on inactive integrins and, together with prior findings, suggest that this interaction restricts alphavbeta3 functions that regulate vascular permeability. Our findings suggest mechanisms for viruses to direct hemorrhagic or vascular diseases and provide a distinct target for modulating alphavbeta3-integrin functions.

Tyrosine residues direct the ubiquitination and degradation of the NY-1 hantavirus G1 cytoplasmic tail.

The hantavirus G1 protein contains a long C-terminal cytoplasmic tail of 142 residues. Hantavirus pulmonary syndrome-associated hantaviruses contain conserved tyrosine residues near the C terminus of G1 which form an immunoreceptor tyrosine activation motif (ITAM) and interact with Src and Syk family kinases. During studies of the G1 ITAM we observed that fusion proteins containing the G1 cytoplasmic tail were poorly expressed. Expression of G1 cytoplasmic tail constructs were dramatically enhanced by treating cells with the proteasome inhibitor ALLN, suggesting that the protein is ubiquitinated and degraded via the 26S proteasome. By using a 6-His-tagged ubiquitin, we demonstrated that the G1 cytoplasmic tail is polyubiquitinated and degraded in the absence of proteasome inhibitors. Expression of only the ITAM-containing domain also directed protein ubiquitination and degradation in the absence of upstream residues. Deleting the C-terminal 51 residues of G1, including the ITAM, stabilized G1 and blocked polyubiquitination and degradation of the protein. Site-directed mutagenesis of both ITAM tyrosines (Y619 and Y632) to phenylalanine also blocked polyubiquitination of G1 proteins and dramatically enhanced G1 protein stability. In contrast, the presence of Y627, which is not part of the ITAM motif, had no effect on G1 stability. Mutagenesis of just Y619 enhanced G1 stability, inhibited G1 ubiquitination, and increased the half-life of G1 by threefold. Mutating only Y632 had less of an effect on G1 protein stability, although Y619 and Y632 synergistically contributed to G1 instability. These findings suggest that Y619, which is conserved in all hantaviruses, is the primary signal for directing G1 ubiquitination and degradation. Collectively these findings indicate that specific conserved tyrosines within the G1 cytoplasmic tail direct the polyubiquitination and degradation of expressed G1 proteins and provide a potential means for down-regulating hantavirus G1 surface glycoproteins and cellular proteins that interact with G1.

Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements.

Hantaviruses infect human endothelial and immune cells, causing two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We have identified key signaling elements termed immunoreceptor tyrosine-based activation motifs (ITAMs) within the G1 cytoplasmic tail of all HPS-causing hantaviruses. ITAMs direct receptor signaling within immune and endothelial cells and the presence of ITAMs in all HPS-causing hantaviruses provides a means for altering normal cellular responses which maintain vascular integrity. The NY-1 G1 ITAM was shown to coprecipitate a complex of phosphoproteins from cells, and the G1 ITAM is a substrate for the Src family kinase Fyn. The hantavirus ITAM coprecipitated Lyn, Syk, and ZAP-70 kinases from T or B cells, while mutagenesis of the ITAM abolished these interactions. In addition, G1 ITAM tyrosines directed intracellular interactions with Syk by mammalian two-hybrid analysis. These findings demonstrate that G1 ITAMs bind key cellular kinases that regulate immune and endothelial cell functions. There is currently no means for establishing the role of the G1 ITAM in hantavirus pathogenesis. However, the conservation of G1 ITAMs in all HPS-causing hantaviruses and the role of these signaling elements in immune and endothelial cells suggest that functional G1 ITAMs are likely to dysregulate normal immune and endothelial cell responses and contribute to hantavirus pathogenesis.

Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses.

Hantaviruses cause two human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantaviruses infect human endothelial cells but cause little or no damage to the infected endothelium. We analyzed with Affymetrix DNA Arrays (Santa Clara, CA) the endothelial cell transcriptional responses directed by hantaviruses associated with HPS [New York-1 virus (NY-1V)], HFRS [Hantaan virus (HTNV)], or by a hantavirus not associated with human disease [Prospect Hill virus (PHV)]. Hantavirus infections induced 117 cellular genes and repressed 25 genes by >3-fold, 4 days postinfection (p.i.). Although >80% of cells were infected by each virus 1 day p.i., PHV induced or repressed 67 genes at this early time compared with three genes altered by HTNV or NY-1V. The early high-level induction of 24 IFN-stimulated genes by PHV (4- to 229-fold) represents a fundamental difference in the temporal regulation of cellular responses by pathogenic and nonpathogenic hantaviruses. Because all hantaviruses induced >23 IFN-stimulated genes at late times p.i., pathogenic hantaviruses appear to suppress early cellular IFN responses that are activated by nonpathogenic hantaviruses. At late times p.i., 13 genes were commonly induced by HTNV and NY-1V that were not induced by PHV. In contrast to NY-1V, HTNV uniquely induced a variety of chemokines and cell adhesion molecules (i.e., IL-8, IL-6, GRO-beta, ICAM), as well as two complement cascade-associated factors that may contribute to immune components of HFRS disease. NY-1V failed to induce most cellular chemokines directed by HTNV (3/14) or genes primarily activated by NF-kappaB. However, NY-1V uniquely induced beta3 integrin-linked potassium channels, which could play a role in HPS-associated vascular permeability. These studies provide a basic understanding of hantavirus-directed cellular responses that are likely to differentiate pathogenic and nonpathogenic hantaviruses, contribute to HFRS and HPS pathogenesis, and provide insight into disease mechanisms and potential therapeutic interventions.