Inhibition of mTORC1 inhibits lytic replication of Epstein-Barr virus in a cell-type specific manner
© Adamson et al.; licensee BioMed Central Ltd. 2014
Received: 2 April 2014
Accepted: 5 June 2014
Published: 11 June 2014
Epstein-Barr virus is a human herpesvirus that infects a majority of the human population. Primary infection of Epstein-Barr virus (EBV) causes the syndrome infectious mononucleosis. This virus is also associated with several cancers, including Burkitt’s lymphoma, post-transplant lymphoproliferative disorder and nasopharyngeal carcinoma. As all herpesvirus family members, EBV initially replicates lytically to produce abundant virus particles, then enters a latent state to remain within the host indefinitely.
Through a genetic screen in Drosophila, we determined that reduction of Drosophila Tor activity altered EBV immediate-early protein function. To further investigate this finding, we inhibited mTOR in EBV-positive cells and investigated subsequent changes to lytic replication via Western blotting, flow cytometry, and quantitative PCR. The student T-test was used to evaluate significance.
mTOR, the human homolog of Drosophila Tor, is an important protein at the center of a major signaling pathway that controls many aspects of cell biology. As the EBV immediate-early genes are responsible for EBV lytic replication, we examined the effect of inhibition of mTORC1 on EBV lytic replication in human EBV-positive cell lines. We determined that treatment of cells with rapamycin, which is an inhibitor of mTORC1 activity, led to a reduction in the ability of B cell lines to undergo lytic replication. In contrast, EBV-positive epithelial cell lines underwent higher levels of lytic replication when treated with rapamycin.
Overall, the responses of EBV-positive cell lines vary when treated with mTOR inhibitors, and this may be important when considering such inhibitors as anti-cancer therapeutic agents.
Epstein-Barr virus (EBV) is a human herpesvirus which has infected a large majority of the world’s human population. This virus infects epithelial cells of the nasopharynx, where it replicates in a lytic or productive manner, as well as B lymphocytes, where the virus enters a latent state . EBV is associated with a plethora of diseases, including infectious mononucleosis, lymphomas (Burkitt’s lymphoma, Hodgkins lymphoma, post-transplant lymphoproliferative disorder), epithelial-based cancers (including nasopharyngeal carcinoma), multiple sclerosis, and the rare but deadly X-linked lymphoproliferative syndrome . The prevalence of this virus, along with its potential for serious disease, necessitate the study of means to inhibit lytic replication, as well as treatments to kill EBV-positive cancer cells.
EBV lytic replication commences upon infection of new cells, or upon reactivation of the latent virus within cells. During lytic replication the first wave of EBV protein activity includes the immediate-early BZLF1 (Z) and BRLF1 (R) proteins. The Z and R proteins are both absolutely necessary for lytic replication to proceed. These are transcription factors that activate transcription from the promoters of the next wave of EBV genes, the early genes. The early genes encode proteins that act to replicate the viral genome. Lastly, the late genes are expressed to provide the virion structural elements .
The Z and R proteins act not only as transcriptional activators, but also physically interact with several cellular proteins including CREB-binding protein (CBP), in order to promote viral replication in lieu of cellular activities [3, 4]. Z and R have also been found to activate MAPK pathways, including the p38, JNK, and ERK pathways [5–7]. Activation of these pathways has been found to be essential for EBV lytic replication. In addition, both Z and R are SUMO-1 modified, which negatively affects Z transcriptional activity, while enhancing R transcriptional activity [8–11]. A potential means of inhibiting viral replication in cells would be to suppress the activity of Z and/or R. Hindering key viral protein/cellular protein interactions may lead to such suppression of Z and R activities, and therefore inhibit EBV lytic replication.
mTOR is a kinase at the heart of a major signaling pathway. mTOR can be found in two different protein complexes, mTORC1 and mTORC2 [12, 13]. Extracellular signals such as various nutrient levels and growth factors impinge on the mTORC1 pathway to control a variety of processes including protein translation, autophagy, cell growth, and mitochondrial metabolism [12–14]. Pathways that activate mTORC1 include the MAPK ERK and Akt signaling pathways, both of which can be activated by phosphatidylinositol 3 kinase (PI3 kinase) . As part of the mTORC1 complex, mTOR promotes the phosphorylation of downstream targets including p70S6K and 4EBP1. These events promote ribosome biogenesis and cap-dependent translation, respectively [13, 15, 16].
Rapamycin is a specific inhibitor of mTOR activity within mTORC1. Rapamycin, also an immunosuppressant, complexes with the protein FKBP-12; this complex then binds to mTOR and inhibits its kinase activity . As inhibition of mTOR subsequently inhibits protein translation and cell growth, rapamycin is an excellent candidate for anti-tumor treatment. In fact rapamycin (or similar mTOR inhibitors) has gained interest for the treatment of cancers, including EBV-associated post-transplant lymphoproliferative disease [17–19].
Previous studies have shown that inhibition of mTOR by treatment with rapamycin is effective in inhibiting Kaposi’s sarcoma herpesvirus (KSHV) lytic replication . The inhibition of lytic replication appears to be due to the inhibition of translation of the immediate-early protein, RTA . Another mTOR inhibitor, Torin1, was found to inhibit viral replication for the herpesvirus members cytomegalovirus, herpes simplex virus 1, and murine gammaherpesvirus 68 . These three viruses were much less sensitive to rapamycin treatment . In the case of human cytomegalovirus, the inhibition of mTOR did not greatly affect the immediate-early proteins, as for KSHV, but appeared to inhibit downstream replication events .
In a Drosophila model system, we identified Tor as a modifier of Z and R activities. Translating this finding to the context of lytically-replicating EBV, we found that mTORC1 inhibition via rapamycin treatment yielded different effects in B cell versus epithelial cell lines. While rapamycin treatment of EBV-positive B cells inhibited lytic replication, rapamycin treatment increased lytic replication in the EBV-positive epithelial cell lines tested, suggesting that the effects of mTOR inhibition differ greatly, in respect to lytic replication, between different cell types. These effects upon EBV lytic replication appear to be, at least in part, due to differential influences upon Z and R gene expression.
Loss of Tor modified GMR-Z and GMR-R phenotypes in Drosophila
Inhibition of mTOR via rapamycin decreases EBV lytic replication in B cell lines, but not in epithelial cell lines
EBV biology is cell-type specific, as the virus lytically replicates in epithelial cells to produce abundant progeny viral particles, but enters a latent state in B cells, immortalizing those cells to remain a part of those cells indefinitely . To examine the effects of mTOR inhibition on EBV lytic replication in EBV-positive B cells, we treated the latently infected B cell line Raji with a dosage series of rapamycin for 24 hr then disrupted latency in these cells. Western blot analysis was performed for BMRF1, and the resulting BMRF1 levels quantified and standardized relative to tubulin levels (Figure 2A, B). In contrast to the epithelial cells, rapamycin treatment inhibited lytic replication in B cells, in a dose-dependent manner. Additional EBV-positive B cell lines were examined, including P3HR1, Daudi, and EBfaV-GFP (derived from B958), and all showed results similar to Raji, i.e. that rapamycin treatment was effective in inhibiting early lytic replication (Figure 2C). The rapamycin dose most effective in decreasing lytic replication in B cells, without impairing cell growth (5 nM), was also very effective in inhibiting the phosphorylation of p70S6K by mTOR in Raji cells (Figure 2E, lane 2).As for AGS-BDneo, we examined whether a higher dose of rapamycin would be more effective than 5 nM rapamycin at inhibiting early lytic replication in Raji cells. Figure 3B shows that 5 nM was very effective at inhibiting BMRF1 expression (Figure 3B, lane 4), and a higher dose (100 nM) was not more effective (Figure 3B, lane 5).
Treatment of EBV-positive cell lines with rapamycin does not impact cell viability
Short-term effects of mTOR inhibition on EBV lytic replication
For all cell lines tested, treatment with rapamycin 18–24 hr prior to induction yielded the most significant results. Rapamycin treatment at the time of induction (0 hr) yielded a milder change, and treatment 24 hr post-induction yielded little to no change in BMRF1 levels (data not shown).
Z and R transcript and protein levels are altered by rapamycin treatment in a cell-type dependent manner
We discovered that modulation of mTOR activity alters EBV lytic replication, beginning with a genetic screen in Drosophila that demonstrated that loss of Tor enhanced Z activity, and suppressed R activity, in Drosophila tissues (Figure 1). It logically followed that inhibition of mTOR in human cells would alter the lytic replication of EBV, as lytic replication is dependent upon both Z and R activities.We found that the rapamycin-mediated inhibition of mTOR had varying effects upon lytic replication amongst different EBV-positive cell lines. Inhibition of mTOR inhibited lytic replication in B cell lines, while in contrast enhanced lytic replication in epithelial cell lines (Figures 2 and 4). The effects upon lytic replication were not time-dependent, as would be found if the action of rapamycin was negated by a negative feedback loop, as short-term doses of rapamycin yielded the same results as longer-term doses [i.e. in B cells rapamycin inhibited lytic replication at all time points, and in epithelial cells rapamycin enhanced lytic replication at all time points (Figure 6)]. Furthermore, in an attempt to have epithelial cells mimic the B cell response to rapamycin, we treated epithelial cells with higher doses of rapamycin, yet lytic replication in these cells was still enhanced by the higher doses (Figure 3).
As the immediate-early proteins Z and R are responsible for initiating lytic replication, and lytic replication was altered upon rapamycin treatment, it follows that the inhibition of mTOR likely altered these proteins’ levels or functions. In the case of KSHV, Nichols et al. found that treatment of KSHV-positive cells with 12 nM rapamycin inhibited the KSHV immediate-early protein RTA from being translated . This logically led to a loss of lytic replication in these cells . However for other herpesviruses such as human cytomegalovirus, treatment with 20 nM rapamycin or 250 nM Torin1 had little effect upon immediate-early protein levels, yet caused a decrease in the number of viral particles produced via lytic replication . Therefore, it does not appear that inhibition of mTORC1 simply leads to an inhibition of viral protein translation for all viruses. In our study we found that rapamycin treatment actually altered the transcript levels of Z and R, such that in B cells Z and R transcript levels were decreased when lytic replication was induced in the presence of rapamycin, while in epithelial cells Z and R transcript levels were increased when lytic replication was induced in the presence of rapamycin (Figure 7A). The resulting Z and R protein levels correlated with the transcript levels (Figure 7B), and with the amount of Z protein binding to Z-response elements (ZREs; data not shown); this correlates with the levels of lytic replication that ensued in each cell type.
mTOR function is central to many different cellular processes, and viruses have evolved to manipulate the mTOR pathways to aid their replication [32, 33]. Our investigations have provided evidence that EBV replication is dependent upon the mTOR pathway, at least in B cells, and that inhibiting this pathway in B cells greatly limits EBV lytic replication. However direct inhibition of mTOR in epithelial cells has the opposite effect, and enhances EBV lytic replication. These results are important to take into account when considering the use of mTOR inhibitors as anti-viral or anti-cancer therapeutic agents.
Materials and methods
Flies were sputter coated with gold in a Pelco model 3 sputter coater 91000. Images were taken on a Hitachi S-4800 SEM microscope.
D98/HR1, a latently infected, EBV-positive epithelial cell line created by the fusion of HeLa cells with EBV-positive P3HR1 cells were maintained in Ham’s F12 medium supplemented with 10% fetal bovine serum along with penicillin, streptomycin, and fungicide. AGS-BDneo and AGS-BX1 are EBV-positive gastric carcinoma cell lines (gifts of L. Hutt-Fletcher) and were maintained in Ham’s F12 supplemented with 10% fetal bovine serum along with penicillin, streptomycin, fungicide, and 500 μg/ml G418. Raji, Daudi, and P3HR1 (ATCC) are latently infected, EBV-positive B cell lines derived from Burkitt’s lymphoma. EBfaV-GFP is a derivative of the B958 cell line, in which the viral LMP2 gene was replaced by the EGFP gene driven by the CMV immediate-early promoter . All B cell lines were maintained in RPMI supplemented with 10% fetal bovine serum along with penicillin, streptomycin, and fungicide.
Cells were treated with rapamycin (in DMSO, Sigma-Aldrich) for the times indicated. Cells not treated with either compound were treated with the vehicle, DMSO (Sigma-Aldrich). If cells were washed prior to induction, cells had their media removed, were washed one time with 1xPBS, then regular media added back. If unwashed, then the rapamycin or DMSO remained present in the media until cells were harvested.
Disruption of viral latency
B cells were treated with 20 ng/ml TPA and 3 mM sodium butyrate, and epithelial cells were treated with 5 ng/ml TPA and 0.75 mM sodium butyrate (higher doses of these inducing chemicals caused significant cell death of our AGS cell lines) for 24–48 hr as indicated (both from Sigma-Aldrich).
Cells were collected, washed twice with 1xPBS, resuspended in ELB lysis buffer (0.25 M NaCl, 0.1% NP40, 50 mM HEPES pH 7, 5 mM EDTA and protease inhibitors), and sonicated. The lysed cells were centrifuged and the supernatants quantified for protein concentration prior to SDS-PAGE.
Equal amounts of protein (20–40 μg) were loaded onto 10% SDS-PAGE gels and electrophoresed. Proteins were transferred to Immobilon (Millipore) membranes.
Blots were blocked with blocking buffer (5% nonfat dried milk, with 0.1% Tween-20, 1xPBS) for 2 hr to overnight. Primary antibodies: anti-BMRF1 (Capricorn), anti-tubulin (Developmental Hybridoma Bank), anti-R (Argene) and anti-Z (Argene or Santa Cruz) were diluted 1:200 in blocking buffer; anti-phospho-p70S6K and anti-total p70S6K (Cell Signaling) were diluted 1:1000 in blocking buffer; all were incubated with the blots 1 hr to overnight, washed with 0.1% Tween-20 in 1xPBS 3 times for 10 min each, and incubated in secondary antibody (goat-anti-mouse HRP or goat-anti-rabbit-HRP, Jackson Immunoresearch) diluted 1:10,000 in blocking buffer, for 1 hr. Blots were washed as before and visualized with Super Signal (Pierce). Images and quantification were performed with a BioRad gel documentation apparatus.
Viability and cell density testing
Cells were treated with rapamycin for 24 hr then induced. Cells were collected and diluted in Guava ViaCount solution. After a five minute incubation, cell viability and cell density (total cells/ml) were determined by flow cytometry with a Guava InCyte flow cytometer.
Cells were treated as indicated, washed with PBS, and fixed with 60% acetone in PBS for 10 min at 4°. Cells were washed with PBS/0.5% BSA, incubated with anti-BMRF1 (Capricorn) or anti-VCA (Argene) antibodies [1:200 in Incubation mix (0.3% BSA, 5% goat serum, 0.1% Triton X in PBS)] for 1 hr at room temperature, washed with PBS/0.5% BSA, and incubated in donkey-anti-mouse-FITC (1:400 in Incubation mix) (Jackson Immunoresearch) for 1 hr at room temperature. Cells were washed with PBS/0.5% BSA, resuspended in PBS, and analyzed with a Guava EasyCyte flow cytometer (Millipore).
Total RNA from treated cells was isolated with the RNeasy mini kit (QIAGEN). qRT-PCR reactions (20 μL) were performed with the Power SYBR Green RNA-to-CT™ 1-Step kit (Applied Biosystems), 10 ng of total RNA, and 20 pmol of each primer, as per the manufacturer’s directions. Primers used for Z were: 5’-ACTGCTGCAGCACTACCGTGAGGTG-3’ (forward) and 5’-GAAATTTAAGAGATCCTCGTCTAA-3’ (reverse) (creating a 150 bp product). Primers for R were: 5’-ATGAGGCCTAAAAAGGATGGCTT-3’ (forward) and 5’-TGAGGACGTTGCAGTAGTCAG-3’ (reverse) (creating a 100 bp product). Reactions were performed and analyzed with an Applied Biosystems Step One Plus real time PCR machine.
Quantification and statistics
Western blots were quantified with ChemiDoc XRS software (BioRad) and flow cytometry data was quantified with Guava EasyCyte analysis software (Millipore). Experiments were done in triplicate, and the student T-test (two-tailed) used to evaluate significance. A P value <0.05 was considered significant.
This work was supported by NIH grants 1R15AI072699-01 and 1R15AI098015-01A1 to A.L.A.
I would like to thank Dr. Lindsey Hutt-Fletcher for the AGS cell lines, and I would like to thank the following undergraduate students for their assistance with this work: Jason Hansen and Jennifer Awuku.
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