ZIKV induction of tristetraprolin in endothelial and Sertoli cells post-transcriptionally inhibits IFNß/λ expression and promotes ZIKV persistence.

IMPORTANCE: Our findings define a novel role for ZIKV-induced TTP expression in regulating IFNß/IFNλ production in primary hBMECs and Sertoli cells. These cells comprise key physiological barriers subverted by ZIKV to access brain and testicular compartments and serve as reservoirs for persistent replication and dissemination. We demonstrate for the first time that the ARE-binding protein TTP is virally induced and post-transcriptionally regulates IFNß/IFNλ secretion. In ZIKV-infected hBMEC and Sertoli cells, TTP knockout increased IFNß/IFNλ secretion, while TTP expression blocked IFNß/IFNλ secretion. The TTP-directed blockade of IFN secretion permits ZIKV spread and persistence in hBMECs and Sertoli cells and may similarly augment ZIKV spread across IFNλ-protected placental barriers. Our work highlights the importance of post-transcriptional ZIKV regulation of IFN expression and secretion in cells that regulate viral access to protected compartments and defines a novel mechanism of ZIKV-regulated IFN responses which may facilitate neurovirulence and sexual transmission.

Establishment of a CPER reverse genetics system for Powassan virus defines attenuating NS1 glycosylation sites and an infectious NS1-GFP11 reporter virus.

Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long-term neurologic damage. Currently, there are no POWV therapeutics, licensed vaccines, or reverse genetics systems for producing infectious POWVs from recombinant DNA. Using a circular polymerase extension reaction (CPER), we generated recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus. NS1 proteins are highly conserved glycoproteins that regulate replication, spread, and neurovirulence. POWV NS1 contains three putative N-linked glycosylation sites that we modified individually in infectious recLI9 mutants (N85Q, N208Q, and N224Q). NS1 glycosylation site mutations reduced replication kinetics and were attenuated, with 1-2 log decreases in titer. Severely attenuated recLI9-N224Q exhibited a 2- to 3-day delay in focal cell-to-cell spread and reduced NS1 secretion but was lethal when intracranially inoculated into suckling mice. However, footpad inoculation of recLI9-N224Q resulted in the survival of 80% of mice and demonstrated that NS1-N224Q mutations reduce POWV neuroinvasion in vivo. To monitor NS1 trafficking, we CPER fused a split GFP11-tag to the NS1 C-terminus and generated an infectious reporter virus, recLI9-NS1-GFP11. Cells infected with recLI9-NS1-GFP11 revealed NS1 trafficking in live cells and the novel formation of large NS1-lined intracellular vesicles. An infectious recLI9-NS1-GFP11 reporter virus permits real-time analysis of NS1 functions in POWV replication, assembly, and secretion and provides a platform for evaluating antiviral compounds. Collectively, our robust POWV reverse genetics system permits analysis of viral spread and neurovirulence determinants in vitro and in vivo and enables the rational genetic design of live attenuated POWV vaccines. IMPORTANCE Our findings newly establish a mechanism for genetically modifying Powassan viruses (POWVs), systematically defining pathogenic determinants and rationally designing live attenuated POWV vaccines. This initial study demonstrates that mutating POWV NS1 glycosylation sites attenuates POWV spread and neurovirulence in vitro and in vivo. Our findings validate a robust circular polymerase extension reaction approach as a mechanism for developing, and evaluating, attenuated genetically modified POWVs. We further designed an infectious GFP-tagged reporter POWV that permits us to monitor secretory trafficking of POWV in live cells, which can be applied to screen potential POWV replication inhibitors. This robust system for modifying POWVs provides the ability to define attenuating POWV mutations and create genetically attenuated recPOWV vaccines.

Measuring Transendothelial Electrical Resistance (TEER) for Dengue Infection Studies.

A growing body of evidence demonstrates that endothelial cells (ECs) play a prominent role in immune-enhanced pathology seen in dengue virus (DENV) infection that might contribute to vascular permeability and hemorrhagic manifestations in severe dengue cases. However, it remains a question of whether DENV infection of ECs directly causes permeability or if extra-endothelial factors such as immune cell activation or antibody-dependent enhancement (ADE) are required. In this chapter, we detail the measurement of the transendothelial electrical resistance (TEER), a quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial monolayers and show that DENV infection of ECs does not cause endothelial permeability in vitro.

Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Novel infection of pericytes by Andes virus enhances endothelial cell permeability.

Andes Virus (ANDV) non-lytically infects pulmonary microvascular endothelial cells (PMECs) causing a severe capillary leak syndrome termed Hantavirus Pulmonary Syndrome (HPS). Basolaterally, PMECs are in contact with pericytes which play critical roles in regulating PMEC permeability and immune cell recruitment. We discovered that ANDV persistently infects primary human vascular pericytes for up to 9 days, and that PMEC monolayer permeability was increased by supernatants from ANDV-infected pericytes. Pericyte-directed PMEC permeability was consistent with the high-level secretion of the permeability factor VEGF (vascular endothelial growth factor) elicited by ANDV-infected pericytes. These findings suggest that ANDV infection of pericytes augments PMEC permeability and reveal a novel mechanism of pericyte-directed vascular barrier dysfunction that contributes to HPS and provides new therapeutic targets.

Blockade of Autocrine CCL5 Responses Inhibits Zika Virus Persistence and Spread in Human Brain Microvascular Endothelial Cells.

Zika virus (ZIKV) is a neurovirulent flavivirus that uniquely causes fetal microcephaly, is sexually transmitted, and persists in patients for up to 6 months. ZIKV persistently infects human brain microvascular endothelial cells (hBMECs) that form the blood-brain barrier (BBB) and enables viral spread to neuronal compartments. We found that CCL5, a chemokine with prosurvival effects on immune cells, was highly secreted by ZIKV-infected hBMECs. Although roles for CCL5 in endothelial cell (EC) survival remain unknown, the presence of the CCL5 receptors CCR3 and CCR5 on ECs suggested that CCL5 could promote ZIKV persistence in hBMECs. We found that exogenous CCL5 induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in hBMECs and that ERK1/2 cell survival signaling was similarly activated by ZIKV infection. Neutralizing antibodies to CCL5, CCR3, or CCR5 inhibited persistent ZIKV infection of hBMECs. While knockout (KO) of CCL5 failed to prevent ZIKV infection of hBMECs, at 3 days postinfection (dpi), we observed a >90% reduction in ZIKV-infected CCL5-KO hBMECs and a multilog reduction in ZIKV titers. In contrast, the addition of CCL5 to CCL5-KO hBMECs dose-dependently rescued ZIKV persistence in hBMECs. Inhibiting CCL5 responses using CCR3 (UCB35625) and CCR5 (maraviroc) receptor antagonists reduced the number of ZIKV-infected hBMECs and ZIKV titers (50% inhibitory concentrations [IC50s] of 2.5 to 12 µM), without cytotoxicity (50% cytotoxic concentration [CC50] of >80 µM). These findings demonstrate that ZIKV-induced CCL5 directs autocrine CCR3/CCR5 activation of ERK1/2 survival responses that are required for ZIKV to persistently infect hBMECs. Our results establish roles for CCL5 in ZIKV persistence and suggest the potential for CCL5 receptor antagonists to therapeutically inhibit ZIKV spread and neurovirulence. IMPORTANCE Our findings demonstrate that CCL5 is required for ZIKV to persistently infect human brain ECs that normally protect neuronal compartments. We demonstrate that ZIKV-elicited CCL5 secretion directs autocrine hBMEC activation of ERK1/2 survival pathways via CCR3/CCR5, and inhibiting CCL5/CCR3/CCR5 responses prevented ZIKV persistence and spread. Our findings demonstrate that ZIKV-directed CCL5 secretion promotes hBMEC survival and reveals an underlying mechanism of ZIKV pathogenesis and spread. We demonstrate that antagonists of CCR3/CCR5 inhibit ZIKV persistence in hBMECs and provide potential therapeutic approaches for preventing ZIKV persistence, spread, and neurovirulence.

Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA.

Andes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs), causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients, virtually every PMEC is infected; however, the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase RhoA in primary human PMECs, causing VE-cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA but coprecipitates RhoGDI (Rho GDP dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C terminus (residues 69 to 204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N terminus (residues 1 to 69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and vascular endothelial growth factor (VEGF) increase RhoA-induced PMEC permeability by directing protein kinase Cα (PKCα) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF-activated PKCα, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKCα and opposing protein kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV-induced capillary permeability, edema, and HPS. IMPORTANCE HPS-causing hantaviruses infect pulmonary endothelial cells (ECs), causing vascular leakage, pulmonary edema, and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses do not lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia-directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34-phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC-phosphorylated RhoGDI, synergistically enhancing hypoxia-directed RhoA activation and PMEC permeability. Our data suggest inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV-directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.

Recombinant ACE2 Expression Is Required for SARS-CoV-2 To Infect Primary Human Endothelial Cells and Induce Inflammatory and Procoagulative Responses.

SARS-CoV-2 causes COVID-19, an acute respiratory distress syndrome (ARDS) characterized by pulmonary edema, viral pneumonia, multiorgan dysfunction, coagulopathy, and inflammation. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptors to infect and damage ciliated epithelial cells in the upper respiratory tract. In alveoli, gas exchange occurs across an epithelial-endothelial barrier that ties respiration to endothelial cell (EC) regulation of edema, coagulation, and inflammation. How SARS-CoV-2 dysregulates vascular functions to cause ARDS in COVID-19 patients remains an enigma focused on dysregulated EC responses. Whether SARS-CoV-2 directly or indirectly affects functions of the endothelium remains to be resolved and is critical to understanding SARS-CoV-2 pathogenesis and therapeutic targets. We demonstrate that primary human ECs lack ACE2 receptors at protein and RNA levels and that SARS-CoV-2 is incapable of directly infecting ECs derived from pulmonary, cardiac, brain, umbilical vein, or kidney tissues. In contrast, pulmonary ECs transduced with recombinant ACE2 receptors are infected by SARS-CoV-2 and result in high viral titers (∼1 × 107/ml), multinucleate syncytia, and EC lysis. SARS-CoV-2 infection of ACE2-expressing ECs elicits procoagulative and inflammatory responses observed in COVID-19 patients. The inability of SARS-CoV-2 to directly infect and lyse ECs without ACE2 expression explains the lack of vascular hemorrhage in COVID-19 patients and indicates that the endothelium is not a primary target of SARS-CoV-2 infection. These findings are consistent with SARS-CoV-2 indirectly activating EC programs that regulate thrombosis and endotheliitis in COVID-19 patients and focus strategies on therapeutically targeting epithelial and inflammatory responses that activate the endothelium or initiate limited ACE2-independent EC infection.IMPORTANCE SARS-CoV-2 infects pulmonary epithelial cells through ACE2 receptors and causes ARDS. COVID-19 causes progressive respiratory failure resulting from diffuse alveolar damage and systemic coagulopathy, thrombosis, and capillary inflammation that tie alveolar responses to EC dysfunction. This has prompted theories that SARS-CoV-2 directly infects ECs through ACE2 receptors, yet SARS-CoV-2 antigen has not been colocalized with ECs and prior studies indicate that ACE2 colocalizes with alveolar epithelial cells and vascular smooth muscle cells, not ECs. Here, we demonstrate that primary human ECs derived from lung, kidney, heart, brain, and umbilical veins require expression of recombinant ACE2 receptors in order to be infected by SARS-CoV-2. However, SARS-CoV-2 lytically infects ACE2-ECs and elicits procoagulative and inflammatory responses observed in COVID-19 patients. These findings suggest a novel mechanism of COVID-19 pathogenesis resulting from indirect EC activation, or infection of a small subset of ECs by an ACE2-independent mechanism, that transforms rationales and targets for therapeutic intervention.

NS5 Sumoylation Directs Nuclear Responses That Permit Zika Virus To Persistently Infect Human Brain Microvascular Endothelial Cells.

Zika virus (ZIKV) is cytopathic to neurons and persistently infects brain microvascular endothelial cells (hBMECs), which normally restrict viral access to neurons. Despite replicating in the cytoplasm, ZIKV and Dengue virus (DENV) polymerases, NS5 proteins, are predominantly trafficked to the nucleus. We found that a SUMO interaction motif in ZIKV and DENV NS5 proteins directs nuclear localization. However, ZIKV NS5 formed discrete punctate nuclear bodies (NBs), while DENV NS5 was uniformly dispersed in the nucleoplasm. Yet, mutating one DENV NS5 SUMO site (K546R) localized the NS5 mutant to discrete NBs, and NBs formed by the ZIKV NS5 SUMO mutant (K252R) were restructured into discrete protein complexes. In hBMECs, NBs formed by STAT2 and promyelocytic leukemia (PML) protein are present constitutively and enhance innate immunity. During ZIKV infection or NS5 expression, we found that ZIKV NS5 evicts PML from STAT2 NBs, forming NS5/STAT2 NBs that dramatically reduce PML expression in hBMECs and inhibit the transcription of interferon-stimulated genes (ISG). Expressing the ZIKV NS5 SUMO site mutant (K252R) resulted in NS5/STAT2/PML NBs that failed to degrade PML, reduce STAT2 expression, or inhibit ISG induction. Additionally, the K252 SUMOylation site and NS5 nuclear localization were required for ZIKV NS5 to regulate hBMEC cell cycle transcriptional responses. Our data reveal NS5 SUMO motifs as novel NB coordinating factors that distinguish flavivirus NS5 proteins. These findings establish SUMOylation of ZIKV NS5 as critical in the regulation of antiviral ISG and cell cycle responses that permit ZIKV to persistently infect hBMECs.IMPORTANCE ZIKV is a unique neurovirulent flavivirus that persistently infects human brain microvascular endothelial cells (hBMECs), the primary barrier that restricts viral access to neuronal compartments. Here, we demonstrate that flavivirus-specific SIM and SUMO sites determine the assembly of NS5 proteins into discrete nuclear bodies (NBs). We found that NS5 SIM sites are required for NS5 nuclear localization and that SUMO sites regulate NS5 NB complex constituents, assembly, and function. We reveal that ZIKV NS5 SUMO sites direct NS5 binding to STAT2, disrupt the formation of antiviral PML-STAT2 NBs, and direct PML degradation. ZIKV NS5 SUMO sites also transcriptionally regulate cell cycle and ISG responses that permit ZIKV to persistently infect hBMECs. Our findings demonstrate the function of SUMO sites in ZIKV NS5 NB formation and their importance in regulating nuclear responses that permit ZIKV to persistently infect hBMECs and thereby gain access to neurons.

Unique Interferon Pathway Regulation by the Andes Virus Nucleocapsid Protein Is Conferred by Phosphorylation of Serine 386.

Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS) and is the only hantavirus shown to spread person to person and cause a highly lethal HPS-like disease in Syrian hamsters. The unique ability of ANDV N protein to inhibit beta interferon (IFNß) induction may contribute to its virulence and spread. Here we analyzed IFNß regulation by ANDV N protein substituted with divergent residues from the nearly identical Maporal virus (MAPV) N protein. We found that MAPV N fails to inhibit IFNß signaling and that replacing ANDV residues 252 to 296 with a hypervariable domain (HVD) from MAPV N prevents IFNß regulation. In addition, changing ANDV residue S386 to the histidine present in MAPV N or the alanine present in other hantaviruses prevented ANDV N from regulating IFNß induction. In contrast, replacing serine with phosphoserine-mimetic aspartic acid (S386D) in ANDV N robustly inhibited interferon regulatory factor 3 (IRF3) phosphorylation and IFNß induction. Additionally, the MAPV N protein gained the ability to inhibit IRF3 phosphorylation and IFNß induction when ANDV HVD and H386D replaced MAPV residues. Mass spectroscopy analysis of N protein from ANDV-infected cells revealed that S386 is phosphorylated, newly classifying ANDV N as a phosphoprotein and phosphorylated S386 as a unique determinant of IFN regulation. In this context, the finding that the ANDV HVD is required for IFN regulation by S386 but dispensable for IFN regulation by D386 suggests a role for HVD in kinase recruitment and S386 phosphorylation. These findings delineate elements within the ANDV N protein that can be targeted to attenuate ANDV and suggest targeting cellular kinases as potential ANDV therapeutics.IMPORTANCE ANDV contains virulence determinants that uniquely permit it to spread person to person and cause highly lethal HPS in immunocompetent hamsters. We discovered that ANDV S386 and an ANDV-specific hypervariable domain permit ANDV N to inhibit IFN induction and that IFN regulation is directed by phosphomimetic S386D substitutions in ANDV N. In addition, MAPV N proteins containing D386 and ANDV HVD gained the ability to inhibit IFN induction. Validating these findings, mass spectroscopy analysis revealed that S386 of ANDV N protein is uniquely phosphorylated during ANDV infection. Collectively, these findings reveal new paradigms for ANDV N protein as a phosphoprotein and IFN pathway regulator and suggest new mechanisms for hantavirus regulation of cellular kinases and signaling pathways. Our findings define novel IFN-regulating virulence determinants of ANDV, identify residues that can be modified to attenuate ANDV for vaccine development, and suggest the potential for kinase inhibitors to therapeutically restrict ANDV replication.

Zika Virus Persistently Infects and Is Basolaterally Released from Primary Human Brain Microvascular Endothelial Cells.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that has emerged as the cause of encephalitis and fetal microencephaly in the Americas. ZIKV uniquely persists in human bodily fluids for up to 6 months, is sexually transmitted, and traverses the placenta and the blood-brain barrier (BBB) to damage neurons. Cells that support persistent ZIKV replication and mechanisms by which ZIKV establishes persistence remain enigmatic but central to ZIKV entry into protected neuronal compartments. The endothelial cell (EC) lining of capillaries normally constrains transplacental transmission and forms the BBB, which selectively restricts access of blood constituents to neurons. We found that ZIKV (strain PRVABC59) persistently infects and continuously replicates in primary human brain microvascular ECs (hBMECs), without cytopathology, for >9 days and following hBMEC passage. ZIKV did not permeabilize hBMECs but was released basolaterally from polarized hBMECs, suggesting a direct mechanism for ZIKV to cross the BBB. ZIKV-infected hBMECs were rapidly resistant to alpha interferon (IFN-α) and transiently induced, but failed to secrete, IFN-ß and IFN-λ. Global transcriptome analysis determined that ZIKV constitutively induced IFN regulatory factor 7 (IRF7), IRF9, and IFN-stimulated genes (ISGs) 1 to 9 days postinfection, despite persistently replicating in hBMECs. ZIKV constitutively induced ISG15, HERC5, and USP18, which are linked to hepatitis C virus (HCV) persistence and IFN regulation, chemokine CCL5, which is associated with immunopathogenesis, as well as cell survival factors. Our results reveal that hBMECs act as a reservoir of persistent ZIKV replication, suggest routes for ZIKV to cross hBMECs into neuronal compartments, and define novel mechanisms of ZIKV persistence that can be targeted to restrict ZIKV spread.IMPORTANCE ZIKV persists in patients, crossing placental and neuronal barriers, damaging neurons, and causing fetal microencephaly. We found that ZIKV persistently infects brain endothelial cells that normally protect neurons from viral exposure. hBMECs are not damaged by ZIKV infection and, analogous to persistent HCV infection, ZIKV constitutively induces and evades antiviral ISG and IFN responses to continuously replicate in hBMECs. As a result, hBMECs provide a protective niche for systemic ZIKV spread and a viral reservoir localized in the normally protective blood-brain barrier. Consistent with the spread of ZIKV into neuronal compartments, ZIKV was released basolaterally from hBMECs. Our findings define hBMEC responses that contribute to persistent ZIKV infection and potential targets for clearing ZIKV infections from hBMECs. These results further suggest roles for additional ZIKV-infected ECs to facilitate viral spread and persistence in the protected placental, retinal, and testicular compartments.

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.

Dengue Virus NS Proteins Inhibit RIG-I/MAVS Signaling by Blocking TBK1/IRF3 Phosphorylation: Dengue Virus Serotype 1 NS4A Is a Unique Interferon-Regulating Virulence Determinant.

UNLABELLED: Dengue virus (DENV) replication is inhibited by the prior addition of type I interferon or by RIG-I agonists that elicit RIG-I/MAVS/TBK1/IRF3-dependent protective responses. DENV infection of primary human endothelial cells (ECs) results in a rapid increase in viral titer, which suggests that DENV inhibits replication-restrictive RIG-I/interferon beta (IFN-ß) induction pathways within ECs. Our findings demonstrate that DENV serotype 4 (DENV4) nonstructural (NS) proteins NS2A and NS4B inhibited RIG-I-, MDA5-, MAVS-, and TBK1/IKKε-directed IFN-ß transcription (>80%) but failed to inhibit IFN-ß induction directed by STING or constitutively active IRF3-5D. Expression of NS2A and NS4B dose dependently inhibited the phosphorylation of TBK1 and IRF3, which suggests that they function at the level of TBK1 complex activation. NS2A and NS4B from DENV1/2/4, as well as the West Nile virus NS4B protein, commonly inhibited TBK1 phosphorylation and IFN-ß induction. A comparative analysis of NS4A proteins across DENVs demonstrated that DENV1, but not DENV2 or DENV4, NS4A proteins uniquely inhibited TBK1. These findings indicate that DENVs contain conserved (NS2A/NS4B) and DENV1-specific (NS4A) mechanisms for inhibiting RIG-I/TBK1-directed IFN responses. Collectively, our results define DENV NS proteins that restrict IRF3 and IFN responses and thereby facilitate DENV replication and virulence. Unique DENV1-specific NS4A regulation of IFN induction has the potential to be a virulence determinant that contributes to the increased severity of DENV1 infections and the immunodominance of DENV1 responses during tetravalent DENV1-4 vaccination. IMPORTANCE: Our findings demonstrate that NS2A and NS4B proteins from dengue virus serotypes 1, 2, and 4 are inhibitors of RIG-I/MDA5-directed interferon beta (IFN-ß) induction and that they accomplish this by blocking TBK1 activation. We determined that IFN inhibition is functionally conserved across NS4B proteins from West Nile virus and DENV1, -2, and -4 viruses. In contrast, DENV1 uniquely encodes an extra IFN regulating protein, NS4A, that inhibits TBK1-directed IFN induction. DENV1 is associated with an increase in severe patient disease, and added IFN regulation by the DENV1 NS4A protein may contribute to increased DENV1 replication, immunodominance, and virulence. The regulation of IFN induction by nonstructural (NS) proteins suggests their potential roles in enhancing viral replication and spread and as potential protein targets for viral attenuation. DENV1-specific IFN regulation needs to be considered in vaccine strategies where enhanced DENV1 replication may interfere with DENV2-4 seroconversion within coadministered tetravalent DENV1-4 vaccines.

Virus interactions with endothelial cell receptors: implications for viral pathogenesis.

The endothelial lining of the vasculature performs a vital role in maintaining fluid barrier functions despite balancing nutrient and fluid content of tissues, repairing localized damage, coordinating responses of a plethora of factors, immune cells and platelets through a multitude of endothelial cell surface receptors. Viruses that nonlytically cause lethal hemorrhagic or edematous diseases engage receptors on vascular and lymphatic endothelial cells, altering normal cellular responses that control capillary leakage and fluid clearance functions with lethal consequences. Recent studies indicate that receptors directing dengue virus and hantavirus infection of the endothelium contribute to the dysregulation of normal endothelial cell signaling responses that control capillary permeability and immune responses that contribute to pathogenesis. Here we present recent studies of virally altered endothelial functions that provide new insight into targeting barrier functions of the endothelium as a potential therapeutic approach.

An innate immunity-regulating virulence determinant is uniquely encoded by the Andes virus nucleocapsid protein.

UNLABELLED: 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-ß)- 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-ß-, 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-ß induction and provide a rationale for the novel pathogenesis and spread of ANDV. IMPORTANCE: 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.

Hantavirus GnT elements mediate TRAF3 binding and inhibit RIG-I/TBK1-directed beta interferon transcription by blocking IRF3 phosphorylation.

Hantaviruses successfully replicate in primary human endothelial cells by restricting the early induction of beta interferon (IFN-ß) 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-ß induction. Here, we define GnT interactions and residues required to inhibit TRAF3-TBK1-directed IFN-ß 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-ß 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-ß 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-ß induction and define virulence determinants within GnTs that may permit the attenuation of pathogenic hantaviruses. IMPORTANCE These findings provide a mechanism for selected hantavirus GnT interactions to regulate RIG-I/TBK1 signaling responses required for IFN-ß induction by inhibiting TBK1 phosphorylation of IRF3. These studies culminate in showing that a single GnT residue, Y627, is required for the NY-1V GnT to inhibit RIG-I/TBK1-directed IRF3 phosphorylation and IFN-ß induction. These findings define a potential virulence determinant within the NY-1V GnT that may permit hantavirus attenuation.

Hantavirus interferon regulation and virulence determinants.

Hantaviruses predominantly replicate in primary human endothelial cells and cause 2 diseases characterized by altered barrier functions of vascular endothelium. Most hantaviruses restrict the early induction of interferon-ß (IFNß) and interferon stimulated genes (ISGs) within human endothelial cells to permit their successful replication. PHV fails to regulate IFN induction within human endothelial cells which self-limits PHV replication and its potential as a human pathogen. These findings, and the altered regulation of endothelial cell barrier functions by pathogenic hantaviruses, suggest that virulence is determined by the ability of hantaviruses to alter key signaling pathways within human endothelial cells. Our findings indicate that the Gn protein from ANDV, but not PHV, inhibits TBK1 directed ISRE, kB and IFNß induction through virulence determinants in the Gn cytoplasmic tail (GnT) that inhibit TBK1 directed IRF3 phosphorylation. Further studies indicate that in response to hypoxia induced VEGF, ANDV infection enhances the permeability and adherens junction internalization of microvascular and lymphatic endothelial cells. These hypoxia/VEGF directed responses are rapamycin sensitive and directed by mTOR signaling pathways. These results demonstrate the presence of at least two hantavirus virulence determinants that act on endothelial cell signaling pathways: one that regulates antiviral IFN signaling responses, and a second that enhances normal hypoxia-VEGF-mTOR signaling pathways to facilitate endothelial cell permeability. These findings suggest signaling pathways as potential targets for therapeutic regulation of vascular deficits that contribute to hantavirus diseases and viral protein targets for attenuating pathogenic hantaviruses.

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.

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.

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.