key: cord-0879348-d5a90dnq
authors: Linero, Florencia N.; Fernández Bell-Fano, Pablo M.; Cuervo, Eugenia; Castilla, Viviana; Scolaro, Luis A.
title: Inhibition of the PI3K/Akt pathway by Ly294002 does not prevent establishment of persistent Junín virus infection in Vero cells
date: 2014-12-07
journal: Arch Virol
DOI: 10.1007/s00705-014-2298-6
sha: d6580eec2b617690edcfa198a093c98bffdb33a2
doc_id: 879348
cord_uid: d5a90dnq

In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNV-infected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.

In previous work, we demonstrated that the arenavirus Junín virus (JUNV) is able to activate Akt by means of the phosphatidylinositol-3-kinase (PI3K) survival pathway during virus entry. This work extends our study, emphasizing the relevance of this pathway in the establishment and maintenance of persistent infection in vitro. During the course of infection, JUNV-infected Vero cells showed a typical cytopathic effect that may be ascribed to apoptotic cell death. Treatment of infected cultures with Ly294002, an inhibitor of the PI3K/Akt pathway, produced an apoptotic response similar to that observed for uninfected cells treated with the drug. This result suggests that virus-induced activation of the PI3K/Akt pathway does not deliver a strong enough anti-apoptotic signal to explain the low proportion of apoptotic cells observed during infection. Also, inhibition of the PI3K/Akt pathway during the acute stage of infection did not prevent the establishment of persistence. Furthermore, treatment of persistently JUNVinfected cells with Ly294002 did not alter viral protein expression. These findings indicate that despite the positive modulation of the PI3/Akt pathway during Junín virus entry, this would not play a critical role in the establishment and maintenance of JUNV persistence in Vero cells.

The arenavirus Junín virus (JUNV), the etiological agent of Argentine hemorrhagic fever, is able to activate serine/ threonine-specific protein kinase B (PKB), also known as Akt, by means of the phosphatidylinositol 3-kinase (PI3K) pathway during virus entry [1] . This early activation, which plays an important role in JUNV uptake, might eventually activate different signaling pathways, contributing to cell survival and, subsequently, virus replication. One of the targets for Akt activity is the eukaryotic initiation factor 4E binding protein (eIF4EBP), which, in its phosphorylated state, releases eIF4E, allowing capdependent translation. However, in the case of JUNV, eIF4E seems to be dispensable for translation of viral proteins, suggesting that this pathway is not relevant for this function [2] . On the other hand, it has been demonstrated that many viruses are able to modulate the PI3K/ Akt pathway, either by its activation or by its inhibition, in order to facilitate viral replication [3] . Activation of Akt has been reported to be associated with blockage of apoptosis, leading to long-term cellular survival, which allows virus replication and viral protein synthesis [4, 5] and, in some cases, oncogenic transformation, as described for polyomaviruses and human papillomaviruses [6, 7] . Similarly, SARS coronavirus (SARS-CoV) induces weak activation of Akt that is not strong enough to impair apoptosis in response to infection but is nevertheless crucial for the establishment of persistence [8, 9] . In contrast, early activation of Akt by respiratory syncytial virus [10] , porcine circovirus type 2 [11] , dengue virus (DENV) [12] or influenza virus [13] is enough to prevent infected cells from early apoptotic cell death. This work extends our study about the participation of PI3K/Akt in JUNV replication, emphasizing the relevance of Akt in establishment and maintenance of persistent JUNV infection in Vero cells.

Data published recently demonstrated that in vitro JUNV infection leads to apoptotic cell death [14] . We therefore analyzed cell survival in cultures infected with JUNV in order to evaluate if activation of PI3K/Akt during virus entry is sufficient to trigger an anti-apoptotic response favoring virus replication. For this purpose, Vero cells were mock infected or infected with JUNV, and at different times postinfection (p.i.), cell viability was measured using an MTT assay, and nuclear fragmentation associated with chromatin condensation as an apoptotic marker was analyzed by the Hoechst technique. Briefly, cell cultures grown on coverslips were fixed with methanol at -20°C for 15 min. The methanol was then discarded, and cultures were dried at room temperature. Cells were stained with Hoechst 33342 (5 lg/ml, 20 min) and observed by fluorescence microscopy. As shown in Fig. 1a , the number of apoptotic cells that could be observed in mock-infected cultures was negligible (dark grey bars), and the proportion of apoptotic cells (light grey bars) detected in infected cultures was modest. The viability of JUNV-infected cells (Fig. 1b , light grey bars) remained practically unchanged throughout the experiment when compared to the value obtained at 24 h p.i., whereas mock-infected cells (Fig. 1b , dark grey bars) grew steadily over time. Detection of apoptotic cells was coincident with the appearance of a distinctive cytopathic effect (CPE), characterized by cell rounding and detachment, that could be readily observed from the third day p.i. onwards (data not shown). Our next approach was to evaluate the participation of the PI3K/Akt pathway in the modulation of the apoptotic state of infected cells. For this purpose, cellular viability and apoptosis were examined in JUNV-infected cells in the presence of the PI3K inhibitor Ly294002 at 72 h p.i. As shown in Fig. 1c , treatment with Ly294002 induced a similar dose-dependent reduction in cell growth for both mock-and JUNV-infected cells. At the same time, JUNVinfected cells treated with Ly294002 showed a larger number of apoptotic cells than did uninfected treated controls (Fig. 1d) . However, when the increase in apoptosis was normalized to the corresponding untreated control (0 lM), the percentage values were similar for both mockand JUNV-infected cultures (Fig. 1d ). In view of these results, it may be concluded that the increase in the number of apoptotic cells in JUNV-infected cultures treated with the drug might have been due to an additive effect between virus and Ly294002. To test this hypothesis, we performed a similar experiment with dengue virus serotype 2 (DENV-2), a virus that positively modulates the PI3K/Akt pathway in order to delay the apoptotic response [12] . As shown in Fig. 1e , inhibition of PI3K with Ly294002 in Vero cells infected with DENV resulted in a large increase in the number of apoptotic cells compared with the mock-infected control. Based on these results, activation of PI3K/Akt triggered by JUNV during entry appears not to be a key factor contributing to the modulation of the apoptotic process in infected cells, suggesting that JUNV is not able to counteract apoptosis via upregulation of the PI3K/Akt pathway. This was confirmed when cells were treated with staurosporine (STS), a multi-factorial strong apoptosis inducer. The proportion of cells showing chromatin condensation and nuclear fragmentation was markedly higher for infected cells than for mock-infected cultures (Fig. 1f) .

Arenaviruses are able to establish persistent infections in vivo, in their natural hosts, as well as in vitro, in several types of cell cultures. Persistence in vivo is primarily governed by suppression of the immune response. This persistence depends on the long-term infection of fibroblastic reticular cells, which are the primary site of virus replication [15] . Viral targeting of fibroblastic reticular cells contributes to immunosuppression, with PDL-1 and IL-10 being the main effectors for suppression [16, 17] .

Thus, looking at long-term infection of a fibroblastic cell line, i.e., Vero cells, is biologically relevant as an approach for understanding in vivo persistence.

In view of the results described above, although JUNV modulation of apoptosis via PI3K/Akt appears not to be relevant for survival of the infected cultures, it might be necessary for the establishment of persistence in vitro. Thus, our next goal was to investigate the participation of this pathway in the establishment of persistent JUNV infection of Vero cells. To this end, JUNV infection was initiated in the absence or in the presence of Ly294002. Cells infected with JUNV at an MOI of 1 pfu/cell were treated with Ly294002 during the first (0-7), second (7) (8) (9) (10) (11) (12) (13) (14) , third (14) (15) (16) (17) (18) (19) (20) (21) or fourth week (21-28) p.i. or left untreated. The progression of infection was monitored by measuring the expression of the JUNV nucleoprotein N and the production of infectious viruses. All of the cultures were able to recover from the CPE observed during the acute stage of infection, and by day 28 p.i., they became morphologically indistinguishable from uninfected Vero cells (data not shown). As shown in Fig. 2a , treatment of infected cells with Ly294002 altered the pattern of expression of N during the course of infection, in contrast with untreated cultures, in which the protein was steadily synthesized throughout the experiment. Inhibition of virus replication by Ly294002 may be ascribed to the effect of the drug on the adsorption/internalization step. Under these conditions (infection at low MOI), the expression of N is the result of multi-step growth, where adsorption/internalization of progeny virus produced after each cycle is hampered by the drug. On the other hand, treatment with inhibitor was not able to abolish productive infection, although infectivity levels in supernatants were higher for cultures treated during the first, second, and third week p.i. (Fig. 2b) . It is tempting to speculate that inhibition of PI3K/Akt would hamper virus replication, thus delaying establishment of persistence. However, it cannot be ruled out that the higher titers detected in treated cultures were the result of the cyclic production of infectivity, which is typical of the early stage of persistence [18, 19] . As shown in Fig. 2c, at 70 days p. i., all cultures synthesized N protein and conserved their capacity to produce infectious virus (Fig. 2b) . In fact, an indirect immunofluorescence assay (IFA) confirmed a similar pattern of expression of N as well as the main envelope glycoprotein (G1) in all cultures (Fig. 2d) . Characterization was continued up to 120 days p.i., at which time persistently JUNV-infected Vero cells, named V3, became non-virogenic although continuing to express high levels of N. Of note, infectivity levels at 70 days p.i. were higher for treated cultures, whereas at 21-28 days p.i., treated cultures showed a low level of infectivity, similar to that of untreated controls, suggesting that treatment with Ly294002 is effective up to the third week of infection. This time point is generally accepted as the transition of acute to persistent JUNV infection in Vero cells [18, 19] . At 120 days p.i., all of the cultures expressed high amounts of N, as shown by Western blot (WB) (Fig. 2e) and IFA (Fig. 2f) . However, as expected, no infectivity could be recovered from them. In agreement with this observation, G1 expression was not detected (data not shown); however, all cultures showed the capacity to form syncytia upon acidification (Fig. 2f) , as described previously [19] . Akt activation has been proposed to contribute to the establishment of persistent infection by DNA viruses such as murine gammaherpesvirus 68 and human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus [20] as well as by an RNA virus, Sindbis virus [21] . For these viruses, persistent infection is promoted by the negative regulation of the lytic infection exerted by the activation of Akt. Our results indicate that inhibition of Akt would not impair the establishment of persistent JUNV infection in Vero cells because all cultures treated with Ly294002 evolved towards persistence in a fashion similar to that observed for untreated cultures.

Finally, we investigated whether the PI3K/Akt pathway is needed in the maintenance of persistence. For this purpose, we evaluated the effect of Ly294002 treatment on the viability of V3 cells. Cells were treated with 10 and 30 lM of Ly294002, and at 24 and 48 h post-treatment, cell viability and cell morphology were evaluated by an MTT assay and phase contrast microscopy, respectively. As shown in Fig. 3a , Ly294002 treatment did not significantly reduce the viability or alter the morphology of V3 cells, whereas Vero cells showed reduced survival and altered morphology when compared to untreated controls, suggesting that persistent cells are more resistant to treatment with this drug. We also evaluated the effect of Ly294002 treatment on JUNV protein synthesis in V3 cells. To this end, V3 cells were treated with Ly294002 and processed for WB. As a control for acutely infected cells, Vero cells were infected with JUNV at an MOI of 1 pfu/cell, and after adsorption, cells were treated with the drug. As shown in Fig. 3b , no alteration in the synthesis of N was observed for V3 cells treated with either concentration of Ly294002, while acutely infected cells showed a dose-dependent reduction in the synthesis of the viral protein (Fig. 3c) , in accordance with previously reported data [1] . Moreover, overexpression of a dominant negative plasmid of Akt [1] did not significantly alter the expression of N in V3 cells analyzed by IFA. In fact, 10 ± 5 % inhibition was observed for V3 cells transfected with the dominant negative plasmid, whereas 87 ± 12 % inhibition was observed for Vero cells transfected and acutely infected with JUNV. These results support the conclusion that activation of Akt would not be a relevant event in V3 cells, not even for an efficient internalization of virus, in view of their non-virogenic phenotype. In order to determine the functionality of Akt in persistently JUNV-infected cells, we initially evaluated the uptake of transferrin (Tf), a ligand that depends on this pathway for the efficient recycling of its receptor. Cells were incubated with TRITC-labeled Tf for 30 min in the presence or absence of 10 lM Ly294002 and were then observed by ultraviolet microscopy. As expected, Vero cells showed a characteristic accumulation of labeled Tf around the nucleus when observed at 30 min after addition of Tf (Fig. 3d, left column) . This accumulation was also observed in V3 cells, although the level of Tf was lower (Fig. 3d, middle column) . Moreover, treatment of V3 cells with Ly294002 induced a scattered pattern of Tf in the cytoplasm, indicating that Tf uptake was efficiently blocked by inhibition of PI3K (Fig. 3d, right  column) . Taking into account that JUNV enters cells by using the Tf receptor, we attempted to determine if superinfection with JUNV is able to induce activation of Akt by phosphorylation at Ser-473 in V3 cells. Serumstarved V3 cells were superinfected with JUNV at an MOI of 1 pfu/cell or mock infected, and at different times postsuperinfection, cells were processed for WB. As shown in Fig. 3e , JUNV superinfection induced a biphasic phosphorylation of Akt, peaking at 15 and 120 min post-viruscell contact, while mock-superinfected V3 cells showed a negligible level of activation. This activation was similar to the biphasic activation reported for acute infection of Vero cells with this virus [1] . Furthermore, Akt phosphorylation induced by JUNV in V3 cells was susceptible to Ly294002 inhibition (Fig. 3f, lane 9 vs. 10) in a fashion similar to that observed for JUNV infection of Vero cells (Fig. 3f, lane 4 vs. lane 5). Likewise, treatment with Ly294002 also reduced the Akt activation induced by insulin in both Vero (Fig. 3f, lane 2 vs. lane 3) and V3 cells (Fig. 3f, lane 7 vs. lane 8). Together, these results suggest that the PI3K-Akt pathway, which is functional in V3 cells, is not involved in the maintenance of long-term persistence.

In this work, we have extended our study of the modulation of the PI3K/Akt signaling pathway during JUNV c Fig. 2 Serum-starved Vero cells grown in a 24-well microtiter plate were infected with JUNV at an MOI of 1 pfu/cell and then left untreated or treated with 10 lM Ly294002 during the first (days 0-7), second (days [7] [8] [9] [10] [11] [12] [13] [14] , third (days [14] [15] [16] [17] [18] [19] [20] [21] or fourth (days 21-28) week p.i. The medium was replaced every 3 days and supernatants and cell lysates were harvested every 7 days up to 28 days p.i. in order to evaluate N protein synthesis (a) and virus yield (b). Cultures were maintained weekly, and evaluation of N protein synthesis was determined at 70 days p.i. (c) and 120 days p.i. (d). Expression of N by IFA and syncytium formation after treatment of cells at pH 5.5 was evaluated at 120 days p.i. (e). Alternatively, expression of the viral antigens N and G1 was evaluated by IFA at 70 days p.i. (f). N and G1 were detected using the monoclonal antibodies NA05AG12 and QB03BE08, respectively [24] . Numbers under the panels indicate N expression, calculated as the ratio of N to actin measured with Image J software. Samples in panel b were analyzed using Student's t-test (*p \ 0.05) infection. We found that modulation of this pathway would not influence the apoptotic response to infection, taking into account that the low level of apoptotic cells in JUNVinfected cultures was not increased by inhibition of this survival pathway. A similar situation has been described for SARS-CoV, where activation of Akt by this virus is not strong enough to prevent viral induction of apoptosis [8] but is strong enough to promote the establishment of a persistent infection [9] . In our case, inhibition of the PI3K/ Akt pathway with Ly294002 was unable to impair the establishment of persistent infection by JUNV in Vero cells. Also, the PI3K/Akt pathway would not be crucial for the maintenance of JUNV persistence, because treatment of V3 cells with Ly294002 neither reduced cell viability nor impaired viral protein synthesis. In the case of measles virus, the downregulation of the PI3K/Akt pathway exerted by this virus has been suggested as a strategy to interfere with T-cell activation during immunosuppression [22] . Downregulation of Akt, either by reducing expression or inhibiting phosphorylation, was not observed during JUNV persistence. In fact, phosphorylation of Akt could be readily observed in V3 cells superinfected with JUNV or stimulated with insulin. Recent findings point out the participation of the Raf/MEK/ERK cellular survival pathway in JUNV replication during the acute stage of infection of Vero cells [23] . It is tempting to postulate a key role of this Serum-starved V3 cells were left uninfected or superinfected with JUNV at an MOI of 1 pfu/cell and at different times p.i. p-Akt (Ser473) was detected by WB (e). Serum-starved Vero and V3 cells were infected with JUNV (MOI 1 pfu/cell) or incubated with insulin (100 nM) in the absence or presence of 20 lM Ly294002. At 15 min post-contact, p-Akt (Ser473) was detected by WB using antibodies from Cell Signalling Technology (f). N was detected using monoclonal antibody NA05AG12 [24] . Numbers under panels indicate the fold change of Akt phosphorylation, calculated as the ratio of p-Akt to Akt-total or of N to actin, measured with Image J software pathway during persistence. Collectively, our results suggest that the PI3K/Akt signaling pathway would not play a critical role in the establishment/maintenance of JUNV persistence in Vero cells.

Participation of the phosphatidylinositol 3-kinase/Akt pathway in Junín virus replication in vitro

Participation of eIF4F complex in Junin virus infection: blockage of eIF4E does not impair virus replication

HijAkt: The PI3K/Akt pathway in virus replication and pathogenesis

Phosphatidylinositol 3-kinase confers resistance to encephalomyocarditis and herpes simplex virus-induced cell death through the activation of distinct downstream effectors

Akt plays a critical role in replication of nonsegmented negative-stranded RNA viruses

Involvement of PP2A in viral and cellular transformation

Human papillomavirus type 16 E7 up-regulates AKT activity through the retinoblastoma protein

Importance of Akt signaling pathway for apoptosis in SARS-CoV-infected Vero E6 cells

JNK and PI3k/Akt signaling pathways are required for establishing persistent SARS-CoV infection in Vero E6 cells

Respiratory syncytial virus inhibits apoptosis and induces NF-jB activity through a phosphatidylinositol 3-kinase-dependent pathway

Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway during porcine circovirus type 2 infection facilitates cell survival and viral replication

Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection

Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling

RIG-I enhanced interferon independent apoptosis upon Junin virus infection

Viral targeting of fibroblastic reticular cells contributes to immunosuppression and persistence during chronic infection

PD-L1 has distinct functions in hematopoietic and nonhematopoietic cells in regulating T cell responses during chronic infection in mice

IL-10 and PD-L1 operate through distinct pathways to suppress T-cell activity during persistent viral infection

Influence of cellular functions on the evolution of persistent infections with Junin virus

Synthesis and expression of viral antigens in Vero cells persistently infected with Junin virus

Inhibition of the phosphatidylinositol 3-kinase-Akt pathway enhances gamma-2 herpesvirus lytic replication and facilitates reactivation from latency

Role for the phosphatidylinositol 3-kinase-Akt-TOR pathway during sindbis virus replication in arthropods

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus

Raf/MEK/ERK pathway activation is required for Junín virus replication

Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses

Inhibition of the PI3K/Akt pathway by Ly294002 475

Acknowledgments This investigation was supported by grants from CONICET (PIP 0443) and UBA (20020110100128BA). LAS is a member of the Research Career CONICET.