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Rapamycin blocks production of KSHV/HHV8: insights into the anti-tumor activity of an immunosuppressant drug.

Nichols LA, Adang LA, Kedes DH - PLoS ONE (2011)

Bottom Line: In latently infected human B cell lines, we found that rapamycin inhibited entry of the virus into the lytic replication cycle, marked by a loss of expression of the lytic switch protein, replication and transcription activator (RTA).To test for viral-specific effects of rapamycin, we focused our studies on a B cell line with resistance to rapamycin-mediated growth inhibition.Using this line, we found that the drug had minimal effect on cell cycle profiles, cellular proliferation, or the expression of other cellular or latent viral proteins, indicating that the RTA suppression was not a result of global cellular dysregulation.

View Article: PubMed Central - PubMed

Affiliation: Myles H. Thaler Center for AIDS and Human Retrovirus Research, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT

Background: Infection with Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) often results in the development of fatal tumors in immunocompromised patients. Studies of renal transplant recipients show that use of the immunosuppressant drug rapamycin, an mTOR inhibitor, both prevents and can induce the regression of Kaposi's sarcoma (KS), an opportunistic tumor that arises within a subset of this infected population. In light of rapamycin's marked anti-KS activity, we tested whether the drug might directly inhibit the KSHV life cycle. We focused on the molecular switch that triggers this predominantly latent virus to enter the lytic (productive) replication phase, since earlier work links this transition to viral persistence and tumorigenesis.

Methods and findings: In latently infected human B cell lines, we found that rapamycin inhibited entry of the virus into the lytic replication cycle, marked by a loss of expression of the lytic switch protein, replication and transcription activator (RTA). To test for viral-specific effects of rapamycin, we focused our studies on a B cell line with resistance to rapamycin-mediated growth inhibition. Using this line, we found that the drug had minimal effect on cell cycle profiles, cellular proliferation, or the expression of other cellular or latent viral proteins, indicating that the RTA suppression was not a result of global cellular dysregulation. Finally, treatment with rapamycin blocked the production of progeny virions.

Conclusions: These results indicate that mTOR plays a role in the regulation of RTA expression and, therefore, KSHV production, providing a potential molecular explanation for the marked clinical success of rapamycin in the treatment and prevention of post-transplant Kaposi's sarcoma. The striking inhibition of rapamycin on KSHV lytic replication, thus, helps explain the apparent paradox of an immunosuppressant drug suppressing the pathogenesis of an opportunistic viral infection.

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Related in: MedlinePlus

Rapamycin inhibits spontaneous and induced RTA expression in a dose-dependent manner regardless of induction pathway.BCBL-1 were treated with 120 nM rapamycin or vehicle for 2 h, and then without (uninduced) or with 0.6 mM VPA (induced). (A) 48 h post-treatment, nuclear extracts were analyzed by immunoblot for RTA expression using the nuclear protein, RCC1, as a loading control. Representative experiment, n = 3. (B) 48 h post-rapamycin, uninduced BCBL-1 cells (top panels) or VPA-induced (bottom panels) were harvested, treated with a dead cell stain, then fixed, stained for intracellular RTA, and analyzed by flow cytometry. Representative plots (n = 7) show RTA expression in live-gated BCBL-1 cells treated with vehicle (left panels) or rapamycin (right panels). (C) Nuclear extracts were analyzed for RTA expression using non-enzymatic infrared detection probes to quantify relative protein levels. Ran (ras-related nuclear protein) was used as loading control. Graph (C, bottom panel) shows immunoblot RTA levels normalized to Ran as a percentage of RTA levels in the vehicle (DMSO) treated control. Representative experiment (n = 2). (D) Induced BCBL-1 cells treated for 48 h with rapamycin at indicated doses were fixed, stained for intracellular RTA and analyzed by flow cytometry. Graph, right, shows percent of RTA+ cells in population indicated by histogram gate. (E) BCBL1 48 h post-treatment cells were harvested and nuclear extracts immunoblotted for RTA and normalized to Ran. Graph shows quantification of bands using non-enzymatic infrared detection probes. Representative experiment (n = 2). (E) BCBL-1 treated with indicated doses of rapamycin and left uninduced (square, dashed line), or induced with either VPA (triangle, solid line), TPA (triangle, dashed line), or CoCl2 (open triangle, solid line) were cultured for 48 hrs in presence of rapamycin, then stained with intracellular RTA. Graph shows percentage of RTA+ cells in live cell population. Mean ± s.e.m.; n for each condition shown in parentheses.
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pone-0014535-g002: Rapamycin inhibits spontaneous and induced RTA expression in a dose-dependent manner regardless of induction pathway.BCBL-1 were treated with 120 nM rapamycin or vehicle for 2 h, and then without (uninduced) or with 0.6 mM VPA (induced). (A) 48 h post-treatment, nuclear extracts were analyzed by immunoblot for RTA expression using the nuclear protein, RCC1, as a loading control. Representative experiment, n = 3. (B) 48 h post-rapamycin, uninduced BCBL-1 cells (top panels) or VPA-induced (bottom panels) were harvested, treated with a dead cell stain, then fixed, stained for intracellular RTA, and analyzed by flow cytometry. Representative plots (n = 7) show RTA expression in live-gated BCBL-1 cells treated with vehicle (left panels) or rapamycin (right panels). (C) Nuclear extracts were analyzed for RTA expression using non-enzymatic infrared detection probes to quantify relative protein levels. Ran (ras-related nuclear protein) was used as loading control. Graph (C, bottom panel) shows immunoblot RTA levels normalized to Ran as a percentage of RTA levels in the vehicle (DMSO) treated control. Representative experiment (n = 2). (D) Induced BCBL-1 cells treated for 48 h with rapamycin at indicated doses were fixed, stained for intracellular RTA and analyzed by flow cytometry. Graph, right, shows percent of RTA+ cells in population indicated by histogram gate. (E) BCBL1 48 h post-treatment cells were harvested and nuclear extracts immunoblotted for RTA and normalized to Ran. Graph shows quantification of bands using non-enzymatic infrared detection probes. Representative experiment (n = 2). (E) BCBL-1 treated with indicated doses of rapamycin and left uninduced (square, dashed line), or induced with either VPA (triangle, solid line), TPA (triangle, dashed line), or CoCl2 (open triangle, solid line) were cultured for 48 hrs in presence of rapamycin, then stained with intracellular RTA. Graph shows percentage of RTA+ cells in live cell population. Mean ± s.e.m.; n for each condition shown in parentheses.

Mentions: Clinical observations suggest that active viral production of KSHV is associated with pathogenesis and that rapamycin inhibits the formation of Kaposi's sarcoma [3], [13], [32]–[36]. Therefore, we next investigated the potential role of mTOR (and its blockade by rapamycin) in lytic replication of the virus. Using RT-PCR, Sin and colleagues found that rapamycin has no discernable effect on baseline levels of viral transcripts among latently infected PEL lines [14]. As a result, we hypothesized that mTOR inhibition may modulate lytic KSHV replication through post-transcriptional effects; therefore, we examined viral protein levels in the presence and absence of mTOR inhibition. As RTA is both necessary and sufficient for the initiation of the lytic cascade, we focused our attention on determining the effects of rapamycin on levels of this critical latent-to-lytic switch protein [5], [6]. After treating BCBL-1 cells with rapamycin for two days, we employed immunoblots to assess relative levels of RTA within nuclear protein extracts, using the nuclear protein RCC1 (regulator of chromosome condensation 1) as a loading control (Figure 2A). We found a low level of spontaneous RTA expression in the vehicle treated BCBL-1 population; however, with rapamycin treatment, RTA became undetectable. We verified this finding using intracellular antibody staining and flow cytometric analysis, observing a greater than 50% reduction in the number of BCBL-1 cells spontaneously expressing RTA at detectable levels (Fig. 2B, top panels). We next tested the ability of rapamycin treatment to suppress RTA levels in the presence of a potent lytic cycle inducing agent, VPA. As we expected, addition of VPA to BCBL-1, in the absence of mTOR inhibition, led to a robust increase in RTA levels by 48 hrs. In stark contrast, pre-treatment with rapamycin almost completely blocked this induction (Fig. 2A). Again, we confirmed this drop in RTA expression by flow cytometric analysis of live cells stained for RTA expression (Fig. 2B, bottom).


Rapamycin blocks production of KSHV/HHV8: insights into the anti-tumor activity of an immunosuppressant drug.

Nichols LA, Adang LA, Kedes DH - PLoS ONE (2011)

Rapamycin inhibits spontaneous and induced RTA expression in a dose-dependent manner regardless of induction pathway.BCBL-1 were treated with 120 nM rapamycin or vehicle for 2 h, and then without (uninduced) or with 0.6 mM VPA (induced). (A) 48 h post-treatment, nuclear extracts were analyzed by immunoblot for RTA expression using the nuclear protein, RCC1, as a loading control. Representative experiment, n = 3. (B) 48 h post-rapamycin, uninduced BCBL-1 cells (top panels) or VPA-induced (bottom panels) were harvested, treated with a dead cell stain, then fixed, stained for intracellular RTA, and analyzed by flow cytometry. Representative plots (n = 7) show RTA expression in live-gated BCBL-1 cells treated with vehicle (left panels) or rapamycin (right panels). (C) Nuclear extracts were analyzed for RTA expression using non-enzymatic infrared detection probes to quantify relative protein levels. Ran (ras-related nuclear protein) was used as loading control. Graph (C, bottom panel) shows immunoblot RTA levels normalized to Ran as a percentage of RTA levels in the vehicle (DMSO) treated control. Representative experiment (n = 2). (D) Induced BCBL-1 cells treated for 48 h with rapamycin at indicated doses were fixed, stained for intracellular RTA and analyzed by flow cytometry. Graph, right, shows percent of RTA+ cells in population indicated by histogram gate. (E) BCBL1 48 h post-treatment cells were harvested and nuclear extracts immunoblotted for RTA and normalized to Ran. Graph shows quantification of bands using non-enzymatic infrared detection probes. Representative experiment (n = 2). (E) BCBL-1 treated with indicated doses of rapamycin and left uninduced (square, dashed line), or induced with either VPA (triangle, solid line), TPA (triangle, dashed line), or CoCl2 (open triangle, solid line) were cultured for 48 hrs in presence of rapamycin, then stained with intracellular RTA. Graph shows percentage of RTA+ cells in live cell population. Mean ± s.e.m.; n for each condition shown in parentheses.
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Related In: Results  -  Collection

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pone-0014535-g002: Rapamycin inhibits spontaneous and induced RTA expression in a dose-dependent manner regardless of induction pathway.BCBL-1 were treated with 120 nM rapamycin or vehicle for 2 h, and then without (uninduced) or with 0.6 mM VPA (induced). (A) 48 h post-treatment, nuclear extracts were analyzed by immunoblot for RTA expression using the nuclear protein, RCC1, as a loading control. Representative experiment, n = 3. (B) 48 h post-rapamycin, uninduced BCBL-1 cells (top panels) or VPA-induced (bottom panels) were harvested, treated with a dead cell stain, then fixed, stained for intracellular RTA, and analyzed by flow cytometry. Representative plots (n = 7) show RTA expression in live-gated BCBL-1 cells treated with vehicle (left panels) or rapamycin (right panels). (C) Nuclear extracts were analyzed for RTA expression using non-enzymatic infrared detection probes to quantify relative protein levels. Ran (ras-related nuclear protein) was used as loading control. Graph (C, bottom panel) shows immunoblot RTA levels normalized to Ran as a percentage of RTA levels in the vehicle (DMSO) treated control. Representative experiment (n = 2). (D) Induced BCBL-1 cells treated for 48 h with rapamycin at indicated doses were fixed, stained for intracellular RTA and analyzed by flow cytometry. Graph, right, shows percent of RTA+ cells in population indicated by histogram gate. (E) BCBL1 48 h post-treatment cells were harvested and nuclear extracts immunoblotted for RTA and normalized to Ran. Graph shows quantification of bands using non-enzymatic infrared detection probes. Representative experiment (n = 2). (E) BCBL-1 treated with indicated doses of rapamycin and left uninduced (square, dashed line), or induced with either VPA (triangle, solid line), TPA (triangle, dashed line), or CoCl2 (open triangle, solid line) were cultured for 48 hrs in presence of rapamycin, then stained with intracellular RTA. Graph shows percentage of RTA+ cells in live cell population. Mean ± s.e.m.; n for each condition shown in parentheses.
Mentions: Clinical observations suggest that active viral production of KSHV is associated with pathogenesis and that rapamycin inhibits the formation of Kaposi's sarcoma [3], [13], [32]–[36]. Therefore, we next investigated the potential role of mTOR (and its blockade by rapamycin) in lytic replication of the virus. Using RT-PCR, Sin and colleagues found that rapamycin has no discernable effect on baseline levels of viral transcripts among latently infected PEL lines [14]. As a result, we hypothesized that mTOR inhibition may modulate lytic KSHV replication through post-transcriptional effects; therefore, we examined viral protein levels in the presence and absence of mTOR inhibition. As RTA is both necessary and sufficient for the initiation of the lytic cascade, we focused our attention on determining the effects of rapamycin on levels of this critical latent-to-lytic switch protein [5], [6]. After treating BCBL-1 cells with rapamycin for two days, we employed immunoblots to assess relative levels of RTA within nuclear protein extracts, using the nuclear protein RCC1 (regulator of chromosome condensation 1) as a loading control (Figure 2A). We found a low level of spontaneous RTA expression in the vehicle treated BCBL-1 population; however, with rapamycin treatment, RTA became undetectable. We verified this finding using intracellular antibody staining and flow cytometric analysis, observing a greater than 50% reduction in the number of BCBL-1 cells spontaneously expressing RTA at detectable levels (Fig. 2B, top panels). We next tested the ability of rapamycin treatment to suppress RTA levels in the presence of a potent lytic cycle inducing agent, VPA. As we expected, addition of VPA to BCBL-1, in the absence of mTOR inhibition, led to a robust increase in RTA levels by 48 hrs. In stark contrast, pre-treatment with rapamycin almost completely blocked this induction (Fig. 2A). Again, we confirmed this drop in RTA expression by flow cytometric analysis of live cells stained for RTA expression (Fig. 2B, bottom).

Bottom Line: In latently infected human B cell lines, we found that rapamycin inhibited entry of the virus into the lytic replication cycle, marked by a loss of expression of the lytic switch protein, replication and transcription activator (RTA).To test for viral-specific effects of rapamycin, we focused our studies on a B cell line with resistance to rapamycin-mediated growth inhibition.Using this line, we found that the drug had minimal effect on cell cycle profiles, cellular proliferation, or the expression of other cellular or latent viral proteins, indicating that the RTA suppression was not a result of global cellular dysregulation.

View Article: PubMed Central - PubMed

Affiliation: Myles H. Thaler Center for AIDS and Human Retrovirus Research, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT

Background: Infection with Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) often results in the development of fatal tumors in immunocompromised patients. Studies of renal transplant recipients show that use of the immunosuppressant drug rapamycin, an mTOR inhibitor, both prevents and can induce the regression of Kaposi's sarcoma (KS), an opportunistic tumor that arises within a subset of this infected population. In light of rapamycin's marked anti-KS activity, we tested whether the drug might directly inhibit the KSHV life cycle. We focused on the molecular switch that triggers this predominantly latent virus to enter the lytic (productive) replication phase, since earlier work links this transition to viral persistence and tumorigenesis.

Methods and findings: In latently infected human B cell lines, we found that rapamycin inhibited entry of the virus into the lytic replication cycle, marked by a loss of expression of the lytic switch protein, replication and transcription activator (RTA). To test for viral-specific effects of rapamycin, we focused our studies on a B cell line with resistance to rapamycin-mediated growth inhibition. Using this line, we found that the drug had minimal effect on cell cycle profiles, cellular proliferation, or the expression of other cellular or latent viral proteins, indicating that the RTA suppression was not a result of global cellular dysregulation. Finally, treatment with rapamycin blocked the production of progeny virions.

Conclusions: These results indicate that mTOR plays a role in the regulation of RTA expression and, therefore, KSHV production, providing a potential molecular explanation for the marked clinical success of rapamycin in the treatment and prevention of post-transplant Kaposi's sarcoma. The striking inhibition of rapamycin on KSHV lytic replication, thus, helps explain the apparent paradox of an immunosuppressant drug suppressing the pathogenesis of an opportunistic viral infection.

Show MeSH
Related in: MedlinePlus