Host mTORC1 Signaling Regulates Andes Virus Replication

Hantavirus pulmonary syndrome (HPS) is a severe respiratory disease characterized by pulmonary edema, with fatality rates of 35 to 45%. Disease occurs following infection with pathogenic New World hantaviruses, such as Andes virus (ANDV), which targets lung microvascular endothelial cells. During replication, the virus scavenges 5′-m⁷G caps from cellular mRNA to ensure efficient translation of viral proteins by the host cell cap-dependent translation machinery. In cells, the mammalian target of rapamycin (mTOR) regulates the activity of host cap-dependent translation by integrating amino acid, energy, and oxygen availability signals. Since there is no approved pharmacological treatment for HPS, we investigated whether inhibitors of the mTOR pathway could reduce hantavirus infection. Here, we demonstrate that treatment with the FDA-approved rapamycin analogue temsirolimus (CCI-779) blocks ANDV protein expression and virion release but not entry into primary human microvascular endothelial cells. This effect was specific to viral proteins, as temsirolimus treatment did not block host protein synthesis. We confirmed that temsirolimus targeted host mTOR complex 1 (mTORC1) and not a viral protein, as knockdown of mTORC1 and mTORC1 activators but not mTOR complex 2 components reduced ANDV replication. Additionally, primary fibroblasts from a patient with tuberous sclerosis exhibited increased mTORC1 activity and increased ANDV protein expression, which were blocked following temsirolimus treatment. Finally, we show that ANDV glycoprotein Gn colocalized with mTOR and lysosomes in infected cells. Together, these data demonstrate that mTORC1 signaling regulates ANDV replication and suggest that the hantavirus Gn protein may modulate mTOR and lysosomal signaling during infection, thus bypassing the cellular regulation of translation.     

Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.     

Andes and Prospect Hill hantaviruses differ in early induction of interferon although both can downregulate interferon signaling

Hantavirus pulmonary syndrome (HPS) is a severe respiratory disease which is thought to result from a dysregulated immune response to infection with pathogenic hantaviruses, such as Sin Nombre virus or Andes virus (ANDV). Other New World hantaviruses, such as Prospect Hill virus (PHV), have not been associated with human disease. Activation of an antiviral state and cell signaling in response to hantavirus infection were examined using human primary lung endothelial cells, the main target cell infected in HPS patients. PHV, but not ANDV, was found to induce a robust beta interferon (IFN-beta) response early after infection of primary lung endothelial cells. The level of IFN induction correlated with IFN regulatory factor 3 (IRF-3) activation, in that IRF-3 dimerization and nuclear translocation were detected in PHV but not ANDV infection. In addition, phosphorylated Stat-1/2 levels were significantly lower in the ANDV-infected cells relative to PHV. Presumably, this reflects the lower level of IRF-3 activation and initial IFN induced by ANDV relative to PHV. To determine whether, in addition, ANDV interference with IFN signaling also contributed to the low Stat-1/2 activation seen in ANDV infection, the levels of exogenous IFN-beta-induced Stat-1/2 activation detectable in uninfected versus ANDV- or PHV-infected Vero-E6 cells were examined. Surprisingly, both viruses were found to downregulate IFN-induced Stat-1/2 activation. Analysis of cells transiently expressing only ANDV or PHV glycoproteins implicated these proteins in this downregulation. In conclusion, while both viruses can interfere with IFN signaling, there is a major difference in the initial interferon induction via IRF-3 activation between ANDV and PHV in infected primary endothelial cells, and this correlates with the reported differences in pathogenicity of these viruses.     

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

A Lethal Disease Model for Hantavirus Pulmonary Syndrome in Immunosuppressed Syrian Hamsters Infected with Sin Nombre Virus

Sin Nombre virus (SNV) is a rodent-borne hantavirus that causes hantavirus pulmonary syndrome (HPS) predominantly in North America. SNV infection of immunocompetent hamsters results in an asymptomatic infection; the only lethal disease model for a pathogenic hantavirus is Andes virus (ANDV) infection of Syrian hamsters. Efforts to create a lethal SNV disease model in hamsters by repeatedly passaging virus through the hamster have demonstrated increased dissemination of the virus but no signs of disease. In this study, we demonstrate that immunosuppression of hamsters through the administration of a combination of dexamethasone and cyclophosphamide, followed by infection with SNV, results in a vascular leak syndrome that accurately mimics both HPS disease in humans and ANDV infection of hamsters. Immunosuppressed hamsters infected with SNV have a mean number of days to death of 13 and display clinical signs associated with HPS, including pulmonary edema. Viral antigen was widely detectable throughout the pulmonary endothelium. Histologic analysis of lung sections showed marked inflammation and edema within the alveolar septa of SNV-infected hamsters, results which are similar to what is exhibited by hamsters infected with ANDV. Importantly, SNV-specific neutralizing polyclonal antibody administered 5 days after SNV infection conferred significant protection against disease. This experiment not only demonstrated that the disease was caused by SNV, it also demonstrated the utility of this animal model for testing candidate medical countermeasures. This is the first report of lethal disease caused by SNV in an adult small-animal model.     

Experimental Andes Virus Infection in Deer Mice: Characteristics of Infection and Clearance in a Heterologous Rodent Host

New World hantaviruses can cause hantavirus cardiopulmonary syndrome with high mortality in humans. Distinct virus species are hosted by specific rodent reservoirs, which also serve as the vectors. Although regional spillover has been documented, it is unknown whether rodent reservoirs are competent for infection by hantaviruses that are geographically separated, and known to have related, but distinct rodent reservoir hosts. We show that Andes virus (ANDV) of South America, carried by the long tailed pygmy rice rat (Oligoryzomys longicaudatus), infects and replicates in vitro and in vivo in the deer mouse (Peromyscus maniculatus), the reservoir host of Sin Nombre virus (SNV), found in North America. In experimentally infected deer mice, viral RNA was detected in the blood, lung, heart and spleen, but virus was cleared by 56 days post inoculation (dpi). All of the inoculated deer mice mounted a humoral immune response by 14 dpi, and produced measurable amounts of neutralizing antibodies by 21 dpi. An up-regulation of Ccl3, Ccl4, Ccl5, and Tgfb, a strong CD4⁺ T-cell response, and down-regulation of Il17, Il21 and Il23 occurred during infection. Infection was transient with an absence of clinical signs or histopathological changes. This is the first evidence that ANDV asymptomatically infects, and is immunogenic in deer mice, a non-natural host species of ANDV. Comparing the immune response in this model to that of the immune response in the natural hosts upon infection with their co-adapted hantaviruses may help clarify the mechanisms governing persistent infection in the natural hosts of hantaviruses.     

Vesicular stomatitis virus-based vaccine protects hamsters against lethal challenge with Andes virus

Andes virus (ANDV) is a highly pathogenic South American hantavirus that causes hantavirus pulmonary syndrome (HPS). A high case fatality rate, the potential for human-to-human transmission, the capacity to infect via aerosolization, and the absence of effective therapies make it imperative that a safe, fast-acting, and effective ANDV vaccine be developed. We generated and characterized a recombinant vesicular stomatitis virus (VSV) vector expressing the ANDV surface glycoprotein precursor (VSVΔG/ANDVGPC) as a possible vaccine candidate and tested its efficacy in the only lethal-disease animal model of HPS. Syrian hamsters immunized with a single injection of VSVΔG/ANDVGPC were fully protected against disease when challenged at 28, 14, 7, or 3 days postimmunization with a lethal dose of ANDV; however, the mechanism of protection seems to differ depending on when the immunization occurs. At 28 days postimmunization, a lack of detectable ANDV RNA in lung, liver, and blood tissue samples, as well as a lack of seroconversion to the ANDV nucleocapsidprotein in nearly all animals, suggested largely sterile immunity. The vaccine was able to generate high levels of neutralizing anti-ANDV G(N)/G(C) antibodies, which seem to play a role as a mechanism of vaccine protection. Administration of the vaccine at 7 or 3 days before challenge also resulted in full protection but with no specific neutralizing humoral immune response, suggesting a possible role of innate responses in protection against challenge virus replication. Administration of the vaccine 24 h postchallenge was successful in protecting 90% of hamsters and again suggested the induction of a potent antiviral state by the recombinant vector as a potential mechanism. Overall, our data suggest the potential for the use of the VSV platform as a fast-acting and effective prophylaxis/postexposure treatment against lethal hantavirus infections.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.     

A Clathrin Independent Macropinocytosis-Like Entry Mechanism Used by Bluetongue Virus-1 during Infection of BHK Cells

Acid dependent infection of Hela and Vero cells by BTV-10 occurs from within early-endosomes following virus uptake by clathrin-mediated endocytosis (Forzan et al., 2007: J Virol 81: 4819–4827). Here we report that BTV-1 infection of BHK cells is also dependent on a low endosomal pH; however, virus entry and infection were not inhibited by dominant-negative mutants of Eps15, AP180 or the ‘aa’ splice variant of dynamin-2, which were shown to inhibit clathrin-mediated endocytosis. In addition, infection was not inhibited by depletion of cellular cholesterol, which suggests that virus entry is not mediated by a lipid-raft dependent process such as caveolae-mediated endocytosis. Although virus entry and infection were not inhibited by the dominant-negative dynamin-2 mutant, entry was inhibited by the general dynamin inhibitor, dynasore, indicating that virus entry is dynamin dependent. During entry, BTV-1 co-localised with LAMP-1 but not with transferrin, suggesting that virus is delivered to late-endosomal compartments without first passing through early-endosomes. BTV-1 entry and infection were inhibited by EIPA and cytochalasin-D, known macropinocytosis inhibitors, and during entry virus co-localised with dextran, a known marker for macropinocytosis/fluid-phase uptake. Our results extend earlier observations with BTV-10, and show that BTV-1 can infect BHK cells via an entry mechanism that is clathrin and cholesterol-independent, but requires dynamin, and shares certain characteristics in common with macropinocytosis.     

Andes Hantavirus-Infection of a 3D Human Lung Tissue Model Reveals a Late Peak in Progeny Virus Production Followed by Increased Levels of Proinflammatory Cytokines and VEGF-A

Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS), a severe acute disease with a 40% case fatality rate. Humans are infected via inhalation, and the lungs are severely affected during HPS, but little is known regarding the effects of ANDV-infection of the lung. Using a 3-dimensional air-exposed organotypic human lung tissue model, we analyzed progeny virus production and cytokine-responses after ANDV-infection. After a 7–10 day period of low progeny virus production, a sudden peak in progeny virus levels was observed during approximately one week. This peak in ANDV-production coincided in time with activation of innate immune responses, as shown by induction of type I and III interferons and ISG56. After the peak in ANDV production a low, but stable, level of ANDV progeny was observed until 39 days after infection. Compared to uninfected models, ANDV caused long-term elevated levels of eotaxin-1, IL-6, IL-8, IP-10, and VEGF-A that peaked 20–25 days after infection, i.e., after the observed peak in progeny virus production. Notably, eotaxin-1 was only detected in supernatants from infected models. In conclusion, these findings suggest that ANDV replication in lung tissue elicits a late proinflammatory immune response with possible long-term effects on the local lung cytokine milieu. The change from an innate to a proinflammatory response might be important for the transition from initial asymptomatic infection to severe clinical disease, HPS.     

Cellular entry of Hantaan virus A9 strain: specific interactions with beta3 integrins and a novel 70kDa protein

Cellular entry of pathogenic hantaviruses had been shown to be mediated by beta3 integrins. However, no direct evidence exists that hantavirus binds to beta3 integrins, and integrin beta3 subunit is not expressed on some cells permissive to hantavirus infection. In this report, utilizing beta3-integrin-transfected CHO cells, we demonstrated that integrin beta3 subunit renders CHO cells susceptible to Chinese Hantaan virus (HTN) strain A9 (isolated in China), and the viral infection was correspondingly inhibited by antibodies to alphavbeta3, alphaIIbbeta3, beta3, and alphav integrins. Furthermore, virus overlay protein-binding assay and 'quarternary Western' analysis indicate that HTN A9 directly interacts with beta3 integrins and an unidentified 70kDa protein. These findings indicate that beta3 integrins play a crucial role in cellular entry of HTN A9 via specific interactions with the virus. In addition, a novel 70kDa protein may serves as a candidate receptor or alternative cellular component for interaction with HTN.     

T cells are not required for pathogenesis in the Syrian hamster model of hantavirus pulmonary syndrome

Andes virus (ANDV) is associated with a lethal vascular leak syndrome in humans termed hantavirus pulmonary syndrome (HPS). In hamsters, ANDV causes a respiratory distress syndrome closely resembling human HPS. The mechanism for the massive vascular leakage associated with HPS is poorly understood; however, T cell immunopathology has been implicated on the basis of circumstantial and corollary evidence. Here, we show that following ANDV challenge, hamster T cell activation corresponds with the onset of disease. However, treatment with cyclophosphamide or specific T cell depletion does not impact the course of disease or alter the number of surviving animals, despite significant reductions in T cell number. These data demonstrate, for the first time, that T cells are not required for hantavirus pathogenesis in the hamster model of human HPS. Depletion of T cells from Syrian hamsters did not significantly influence early events in disease progression. Moreover, these data argue for a mechanism of hantavirus-induced vascular permeability that does not involve T cell immunopathology.     

Human subtilase SKI-1/S1P is a master regulator of the HCV Lifecycle and a potential host cell target for developing indirect-acting antiviral agents

HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1)--or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of novel broad-spectrum biopharmaceuticals that could lead to novel indirect-acting antiviral options with the current standard of care.     

Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC), caused by viruses within human adenovirus species D (HAdV-D), is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD) profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with downstream proinflammatory cell signaling.     

Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

Background

Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process.

Methodology/Principal Findings

We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion.

Conclusions/Significance

Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.     

Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells

Cholesterol represents one of the key constituents of small, dynamic, sterol- and sphingolipid-enriched domains on the plasma membrane. It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection. In this study, the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus (PRRSV) infection of MARC-145 cells was investigated. Pretreatment of MARC-145 cells with methyl-β-cyclodextrin (MβCD), a drug used to deplete cholesterol from cellular membrane, significantly reduced PRRSV infection in a dose-dependent manner. This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment, suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol. Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry, especially virus attachment and release. These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.