Hantaviruses and TNF-alpha act synergistically to induce ERK1/2 inactivation in Vero E6 cells
© Strandin et al; licensee BioMed Central Ltd. 2008
Received: 30 April 2008
Accepted: 29 September 2008
Published: 29 September 2008
We have previously reported that the apathogenic Tula hantavirus induces apoptosis in Vero E6 epithelial cells. To assess the molecular mechanisms behind the induced apoptosis we studied the effects of hantavirus infection on cellular signaling pathways which promote cell survival. We previously also observed that the Tula virus-induced cell death process is augmented by external TNF-α. Since TNF-α is involved in the pathogenesis of hantavirus-caused hemorrhagic fever with renal syndrome (HFRS) we investigated its effects on HFRS-causing hantavirus-infected cells.
We studied both apathogenic (Tula and Topografov) and pathogenic (Puumala and Seoul) hantaviruses for their ability to regulate cellular signaling pathways and observed a direct virus-mediated down-regulation of external signal-regulated kinases 1 and 2 (ERK1/2) survival pathway activity, which was dramatically enhanced by TNF-α. The fold of ERK1/2 inhibition correlated with viral replication efficiencies, which varied drastically between the hantaviruses studied.
We demonstrate that in the presence of a cytokine TNF-α, which is increased in HFRS patients, hantaviruses are capable of inactivating proteins that promote cell survival (ERK1/2). These results imply that hantavirus-infected epithelial cell barrier functions might be compromised in diseased individuals and could at least partially explain the mechanisms of renal dysfunction and the resulting proteinuria seen in HFRS patients.
Hantaviruses (Family Bunyaviridae, Genus Hantavirus) are viruses which chronically infect rodents and insectivores with no apparent disease but in humans they cause two major clinical symptoms: HFRS in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Some hantaviruses also seem to be apathogenic, including Tula (TULV) and Topografov (TOPV) virus [1, 2]. Depending on the causative virus, HFRS manifests as mild (Puumala virus; PUUV), moderate (Seoul virus; SEOV) or severe disease (Hantaan virus; HTNV). Hantaviruses are negative-sense single-stranded RNA viruses with a tripartite genome of large (L), medium (M) and small (S) segments encoding the RNA-dependent RNA polymerase, the envelope precursor protein of two glycoproteins Gn and Gc, and the nucleocapsid protein N .
The multi-organ hantaviral disease is characterized by local induction of cytokines but their role in the mechanisms of pathogenesis is still poorly understood. Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine associated with hantavirus infections in vivo. Elevated TNF-α levels are found in plasma of HFRS [4, 5] and HCPS  patients and TNF-α has been detected directly in the kidneys of NE patients . TNF-α is implicated in the pathophysiology of, for example, septic shock and is capable of inducing adult respiratory distress syndrome (ARDS) in experimental animals and humans. The strong similarity of these effects to the manifestations in hantavirus diseases , together with the evidence of association of TNF-α polymorphism of high-producer haplotype in the severe course of PUUV infection , makes TNF-α a factor in hantavirus pathogenesis which deserves further attention. TNF-a is a conditional death inducer with pro-apoptotic capacity only uncovered when cell survival mechanisms are hindered. TNF-α-induced programmed cell death occurs via the cleavage of procaspase-8 to its active form, thereby initiating the caspase cascade leading to poly ADP-ribose polymerase (PARP) cleavage among others and eventually apoptosis .
Previous work done in our laboratory demonstrated that TULV infection induces apoptosis in Vero E6 cells and that externally added TNF-α enhances the cell death process . To shed light on the molecular mechanisms which facilitate TNF-α mediated apoptosis in hantavirus-infected cells, we studied the activation of extracellular-signal regulated kinases 1 and 2 (collectively referred to as ERK1/2), a well-known group of mitogen-activated kinases (MAPKs) and regulators of cell survival. We now show that both apathogenic and HFRS-causing hantaviruses act in synergy with TNF-α to inactivate the ERK survival pathway.
Results and discussion
TULV inhibits ERK1/2 activity in Vero E6 cells
HFRS-causing hantaviruses do not have the same capability as TULV to inhibit ERK1/2 activity
Hantaviruses and TNF-α act synergistically to inhibit ERK1/2 activity
In characterization of the mechanisms of hantavirus-mediated apoptosis further, we demonstrated virus replication-dependent down-regulation of ERK1/2 by TULV, TOPV and SEOV, which was synergistically enhanced by TNF-α. ERK1/2 inhibition was induced by TNF-α also in PUUV-infected cells. ERK1/2 refers to prototype members of the mitogen-activated protein kinase (MAPK)-family that regulate cell proliferation, cell differentiation, cell cycle and cell survival . ERK1/2 is activated by phosphorylation to threonine and tyrosine residues, which results in ERK1/2 translocation from the cytosol to the nucleus to regulate transcription. The ERK1/2 pathway is activated in many types of cancer and it promotes cell survival, i.e. it induces anti-apoptotic genes such as Bcl-2 and inactivates the pro-apoptotic Bad . In addition, activation of the ERK1/2 pathway has been shown to protect cells from TNF-α-induced apoptosis [14, 15]. ERK1/2 activity has been shown to be required for the efficient replication of many viruses [16–21]. In contrast, some viral proteins, like Ebola virus glycoprotein , hepatitis C virus non-structural protein NS5A , and human immunodeficiency virus (HIV) type 1 vpr protein  have been shown to down-regulate ERK1/2 activity. To our knowledge, however, our results are the first showing a direct virus replication-mediated down-regulation of ERK1/2 survival pathway in cell culture. Our results show a high basal ERK1/2 activity in confluent mock-infected Vero E6 cells that promotes cell survival even in the presence of sustained TNF-α treatment. However, in the infected cells ERK1/2 activity is reduced, which might at least in part render these cells sensitive to external TNF-α-mediated apoptosis. It would be of interest to understand the role of ERK1/2 activity in terms of viability of hantavirus-infected cells in more detail. Whether external activation of this pathway can rescue from hantavirus-mediated cell death remain to be answered.
The first evidence of hantavirus-induced apoptosis in cultured cells was described in Vero E6 cells with Hantaan virus, the prototype hantavirus to cause HFRS, and with Prospect Hill, an apparently apathogenic hantavirus . Vero E6 cells are derived from monkey kidney epithelium and another kidney epithelial cell line, HEK-293, was later also shown to be susceptible to hantavirus-mediated apoptotic cell death . Besides regulating apoptosis, ERK proteins have other essential roles in the kidneys. They promote tubular epithelial cell proliferation [27, 28] and epithelial cell barrier resistance [29, 30] thereby maintaining the integrity of a functional organ. Taken together with our previous work on TULV-induced apoptosis of Vero E6 cells [11, 31] the present findings show that hantaviruses can hazard epithelial cell viability through apoptosis and ERK1/2 inactivation, at least in the presence of TNF-α.
In HFRS, one of the most prominent clinical manifestations is renal dysfunction leading to proteinuria. Kidney tubular epithelium degeneration and tubular epithelial cell death have been suggested to occur in PUUV-caused HFRS . Also, hantaviral antigens have been detected in the renal tubular epithelial cells of HTNV-  and PUUV-infected patients . Although epithelial cells may not be the main site of viral replication in man in the case of HFRS, viral replication in renal tubular epithelial cells could be the direct cause of renal epithelium dysfunction through direct virus-induced inhibition of signaling pathways necessary for cell viability (ERK1/2), which would be amplified by cytokines elevated in HFRS (TNF-α). Interestingly, Klingström et al.  showed recently an increase in the caspase cleavage product CK18, a marker for epithelial cell apoptosis, in sera of patients infected with PUUV. While the apathogen TULV also has the capacity to induce apoptosis and ERK1/2 inactivation in epithelial cells, one might rationalize that due to unidentified viral determinants apathogenic hantaviruses never make contact with the renal epithelium in vivo or are efficiently eliminated without causing notable renal symptoms or disease.
Viruses and cell cultures
TULV Moravia strain 5302, TOPV, SEOV and PUUV Sotkamo strain were propagated in Vero E6 cells in which they have been isolated and to which they are adapted producing titers of 104-107 focus forming units (FFU)/ml conditioned medium [1, 36, 37]. Vero E6 cells (green monkey kidney epithelial cell line; ATCC: CRL-1586) were grown in minimal essential medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 IU/ml of penicillin and 100 μg/ml of streptomycin, at 37°C in a humidified atmosphere containing 5% CO2. For the experiments, Vero E6 cell monolayers were grown to confluence, virus adsorbed for one hour at 37°C and growth medium added. For mock infections, either fresh culture medium or UV-inactivated virus was used. UV-inactivation was achieved using a stock of virus on ice in a lid-less 3 cm diameter culture dish, which was irradiated at 254 nm using a 30 W UV lamp at a distance of 10 cm with an exposure time of 30 min. The medium of infected and mock-infected cultures was changed once a week. In experiments where TNF-α was used, fresh TNF-α was added together with medium change. Viral titers in supernatants of infected cells were determined as described by Kallio et al. . Briefly, 10-fold diluted supernatants were grown in Vero E6 cells on a 10-well microscopic slide and fluorescently stained for virus. Standard deviations were calculated from 4 individual wells. TULV-conditioned medium collected at 7 days p.i. and TOPV-, SEOV- and PUUV-conditioned media collected at 14 days p.i. were stored at -70°C and used as virus inocula.
Antibodies and reagents
Mouse monoclonal antibody against phosphorylated form of ERK1/2 was from Santa Cruz Biotechnology Inc. Mouse monoclonal antibody against cleaved PARP and rabbit polyclonal antibody against ERK1/2 were from Cell Signaling Biotechnology. Rabbit polyclonal antibodies against Puumala hantavirus N have been described previously . Recombinant human TNF-α was from R&D Systems.
Infected and mock-infected Vero E6 cells (grown in 75-cm2 or 25-cm2 flasks) were scraped off into medium, washed twice with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation (RIPA) buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 3 mM EDTA, 1% NP-40, 1 mM dithiothreitol (DTT), 1 mM Na3VO4, 20 mM NaF and EDTA-free cocktail of protease inhibitors (Roche). The protein concentrations of the cell lysates were determined using BCA Protein Assay Kit (Pierce). Laemmli gel loading buffer was added into samples, which were denatured at 95°C for 5 min and stored at -20°C. Samples were analyzed by immunoblotting according to standard protocols using 10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE).
We thank Leena Kostamovaara and Tytti Manni for expert technical assistance. This work was supported by the Academy of Finland grant 102371, EU grant (QLK2-CT-2002-01358), Sigrid Jusélius Foundation, Paulo Foundation, Orion-Farmos Research Foundation, and Finnish Culture Foundation, Helsinki, Finland.
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