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.
endothelial; hantavirus; dengue; arenavirus; PD-L1; platelet; permeability barrier; tolerance induction
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.
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.
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.
Hantaviruses successfully replicate in primary human endothelial cells by restricting the early induction of beta interferon (IFN-β) and interferon-stimulated genes (ISGs). Gn proteins from NY-1V, ANDV, and TULV, but not PHV, harbor elements in their 142-residue cytoplasmic tails (GnTs) that inhibit RIG-I/MAVS/TBK1-TRAF3-directed IFN-β induction. Here, we define GnT interactions and residues required to inhibit TRAF3-TBK1-directed IFN-β induction and IRF3 phosphorylation. We observed that GnTs bind TRAF3 via residues within the TRAF-N domain (residues 392 to 415) and that binding is independent of the MAVS-interactive TRAF-C domain (residues 415 to 568). We determined that GnT binding to TRAF3 is mediated by C-terminal degrons within NY-1V or ANDV GnTs and that mutations that add degrons to TULV or PHV GnTs confer TRAF3 binding. Further analysis of GnT domains revealed that TRAF3 binding is a discrete GnT function, independent of IFN regulation, and that residues 15 to 42 from the NY-1V GnT C terminus are required for inhibiting TBK1-directed IFN-β transcription. Mutagenesis of the NY-1V GnT revealed that altering tyrosine 627 (Y627A/S/F) abolished GnT regulation of RIG-I/TBK1-directed IRF3 phosphorylation and transcriptional responses of ISRE, κB, and IFN-β promoters. Moreover, GnTs from NY-1V, ANDV, and TULV, but not PHV, inhibited RIG-I-directed IRF3 phosphorylation. Collectively, these findings suggest a novel role for GnTs in regulating RIG-I/TBK1 pathway-directed IRF3 phosphorylation and IFN-β induction and define virulence determinants within GnTs that may permit the attenuation of pathogenic hantaviruses.
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.
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 (ANDV) is the only hantavirus known to spread from person to person and shown to cause highly lethal hantavirus pulmonary syndrome (HPS) in patients and Syrian hamsters. Hantaviruses replicate in human endothelial cells and accomplish this by restricting the early induction of beta interferon (IFN-β)- and IFN-stimulated genes (ISGs). Our studies reveal that the ANDV nucleocapsid (N) protein uniquely inhibits IFN signaling responses directed by cytoplasmic double-stranded RNA (dsRNA) sensors RIG-I and MDA5. In contrast, N proteins from Sin Nombre, New York-1, and Prospect Hill hantaviruses had no effect on RIG-I/MDA5-directed transcriptional responses from IFN-β-, IFN-stimulated response element (ISRE)-, or κB-containing promoters. Ablating a potential S-segment nonstructural open reading frame (ORF) (NSs) within the ANDV plasmid expressing N protein failed to alter IFN regulation by ANDV N protein. Further analysis demonstrated that expressing the ANDV N protein inhibited downstream IFN pathway activation directed by MAVS, TBK1, and IκB kinase ε (IKKε) but failed to inhibit transcriptional responses directed by constitutive expression of active interferon regulatory factor IRF3-5D or after stimulation by alpha interferon (IFN-α) or tumor necrosis factor alpha (TNF-α). Consistent with IFN pathway-specific regulation, the ANDV N protein inhibited TBK1-directed IRF3 phosphorylation (phosphorylation of serine 396 [pS396]) and TBK1 autophosphorylation (pS172). Collectively, these findings indicate that the ANDV N inhibits IFN signaling responses by interfering with TBK1 activation, upstream of IRF3 phosphorylation and NF-κB activation. Moreover, our findings reveal that ANDV uniquely carries a gene encoding a virulence determinant within its N protein that is capable of restricting ISG and IFN-β induction and provide a rationale for the novel pathogenesis and spread of ANDV.
Andes virus (ANDV) is distinguished from other hantaviruses by its unique ability to spread from person to person and cause lethal hantavirus pulmonary syndrome (HPS)-like disease in Syrian hamsters. However, virulence determinants that distinguish ANDV from other pathogenic hantaviruses have yet to be defined. Here we reveal that ANDV uniquely contains a virulence determinant within its nucleocapsid (N) protein that potently inhibits innate cellular signaling pathways. This novel function of the N protein provides a new mechanism for hantaviruses to regulate interferon (IFN) and IFN-stimulated gene (ISG) induction that is likely to contribute to the enhanced ability of ANDV to replicate, spread, and cause disease. These findings differentiate ANDV from other HPS-causing hantaviruses and provide a potential target for viral attenuation that needs to be considered in vaccine development.
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.
Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Preexisting antibodies to dengue virus disposes patients to immune-enhanced edema (DSS) or hemorrhagic (DHF) disease following infection by a discrete dengue virus serotype. Although the endothelium is the primary vascular fluid barrier, direct effects of dengue virus on endothelial cells (ECs) have not been considered primary factors in pathogenesis. Here, we show that dengue virus infection of human ECs elicits immune-enhancing EC responses. Our results suggest that rapid early dengue virus proliferation within ECs is permitted by dengue virus regulation of early, but not late, beta interferon (IFN-β) responses. The analysis of EC responses following synchronous dengue virus infection revealed the high-level induction and secretion of immune cells (T cells, B cells, and mast cells) as well as activating and recruiting cytokines BAFF (119-fold), IL-6/8 (4- to 7-fold), CXCL9/10/11 (45- to 338-fold), RANTES (724-fold), and interleukin-7 (IL-7; 128-fold). Moreover, we found that properdin factor B, an alternative pathway complement activator that directs chemotactic anaphylatoxin C3a and C5a production, was induced 34-fold. Thus, dengue virus-infected ECs evoke key inflammatory responses observed in dengue virus patients which are linked to DHF and DSS. Our findings suggest that dengue virus-infected ECs directly contribute to immune enhancement, capillary permeability, viremia, and immune targeting of the endothelium. These data implicate EC responses in dengue virus pathogenesis and further rationalize therapeutic targeting of the endothelium as a means of reducing the severity of dengue virus disease.
Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The endothelium is the primary fluid barrier of the vasculature and ultimately the effects of dengue virus infection that cause capillary leakage impact endothelial cell (EC) barrier functions. The ability of dengue virus to infect the endothelium provides a direct means for dengue to alter capillary permeability, permit virus replication, and induce responses that recruit immune cells to the endothelium. Recent studies focused on dengue virus infection of primary ECs have demonstrated that ECs are efficiently infected, rapidly produce viral progeny, and elicit immune enhancing cytokine responses that may contribute to pathogenesis. Furthermore, infected ECs have also been implicated in enhancing viremia and immunopathogenesis within murine dengue disease models. Thus dengue-infected ECs have the potential to directly contribute to immune enhancement, capillary permeability, viremia, and immune targeting of the endothelium. These effects implicate responses of the infected endothelium in dengue pathogenesis and rationalize therapeutic targeting of the endothelium and EC responses as a means of reducing the severity of dengue virus disease.
Hantaviruses primarily infect human endothelial cells (ECs) and cause two highly lethal human diseases. Early addition of Type I interferon (IFN) to ECs blocks hantavirus replication and thus for hantaviruses to be pathogenic they need to prevent early interferon induction. PHV replication is blocked in human ECs, but not inhibited in IFN deficient VeroE6 cells and consistent with this, infecting ECs with PHV results in the early induction of IFNβ and an array of interferon stimulated genes (ISGs). In contrast, ANDV, HTNV, NY-1V and TULV hantaviruses, inhibit early ISG induction and successfully replicate within human ECs. Hantavirus inhibition of IFN responses has been attributed to several viral proteins including regulation by the Gn proteins cytoplasmic tail (Gn-T). The Gn-T interferes with the formation of STING-TBK1-TRAF3 complexes required for IRF3 activation and IFN induction, while the PHV Gn-T fails to alter this complex or regulate IFN induction. These findings indicate that interfering with early IFN induction is necessary for hantaviruses to replicate in human ECs, and suggest that additional determinants are required for hantaviruses to be pathogenic. The mechanism by which Gn-Ts disrupt IFN signaling is likely to reveal potential therapeutic interventions and suggest protein targets for attenuating hantaviruses.
Dengue virus causes leakage of the vascular endothelium, resulting in dengue hemorrhagic fever and dengue shock syndrome. The endothelial cell lining of the vasculature regulates capillary permeability and is altered by immune and chemokine responses which affect fluid barrier functions of the endothelium. Our findings indicate that human endothelial cells are highly susceptible to infection by dengue virus (type 4). We found that dengue virus productively infects ∼80% of primary human endothelial cells, resulting in the rapid release of ∼105 virions 1 day postinfection. Analysis of potential inhibitors of dengue virus entry demonstrated that antibodies and ligands to integrins and cellular receptors were unable to inhibit dengue virus infection of endothelial cells. In contrast, pretreating cells with heparin or heparan sulfate resulted in a 60 to 80% reduction in dengue virus-infected cells, and pretreatment of endothelial cells with heparinase III or protease reduced dengue infectivity by >80%. Dengue virus bound specifically to resin immobilized heparin, and binding was competitively inhibited by excess heparin but not other ligands. Collectively, these findings suggest that dengue virus specifically attaches to heparan sulfate-containing proteoglycan receptors on endothelial cells. Following attachment to human endothelial cell receptors, dengue virus causes a highly productive infection that has the potential to increase viral dissemination and viremia. This provides the potential for dengue virus-infected endothelial cells to directly alter barrier functions of the endothelium, contribute to enhancement of immune cell activation, and serve as potential targets of immune responses which play a central role in dengue pathogenesis.
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.
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.
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.
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.
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.
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.
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.
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.
The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic α-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 α-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted α-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.
Pathogenic hantaviruses cause two human diseases: hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). The hantavirus G1 protein contains a long, 142-amino-acid cytoplasmic tail, which in NY-1 virus (NY-1V) is ubiquitinated and proteasomally degraded (E. Geimonen, I. Fernandez, I. N. Gavrilovskaya, and E. R. Mackow, J. Virol. 77: 10760-10768, 2003). Here we report that the G1 cytoplasmic tails of pathogenic Andes (HPS) and Hantaan (HFRS) viruses are also degraded by the proteasome and that, in contrast, the G1 tail of nonpathogenic Prospect Hill virus (PHV) is stable and not proteasomally degraded. We determined that the signals which direct NY-1V G1 tail degradation are present in a hydrophobic region within the C-terminal 30 residues of the protein. In contrast to that of PHV, the NY-1V hydrophobic domain directs the proteasomal degradation of green fluorescent protein and constitutes an autonomous degradation signal, or “degron,” within the NY-1V G1 tail. Replacing 4 noncontiguous residues of the NY-1V G1 tail with residues present in the stable PHV G1 tail resulted in a NY-1V G1 tail that was not degraded by the proteasome. In contrast, changing a different but overlapping set of 4 PHV residues to corresponding NY-1V residues directed proteasomal degradation of the PHV G1 tail. The G1 tails of pathogenic, but not nonpathogenic, hantaviruses contain intervening hydrophilic residues within the C-terminal hydrophobic domain, and amino acid substitutions that alter the stability or degradation of NY-1V or PHV G1 tails result from removing or adding intervening hydrophilic residues. Our results identify residues that selectively direct the proteasomal degradation of pathogenic hantavirus G1 tails. Although a role for the proteasomal degradation of the G1 tail in HPS or HFRS is unclear, these findings link G1 tail degradation to viral pathogenesis and suggest that degrons within hantavirus G1 tails are potential virulence determinants.
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.