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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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Virion production is significantly decreased by rapamycin treatment.BCBL-1 +/− rapamycin 12 nM were induced with CoCl2, TPA, or VPA. Five days post-induction, virus was concentrated from supernatants. HeLa cells were then infected with concentrated virus in the presence of polybrene. Viral titers were determined by staining HeLa cells for punctate intranuclear LANA dots and analyzing via multispectral imaging flow cytometry. (A) Images show representative nuclear staining with DRAQ5 (Nuc.), LANA antibody stain, and the overlay (Comb.). Upper panel shows three representative HeLa cells infected with inoculum from untreated VPA-induced BCBL-1. Lower panel shows representative HeLas infected with inoculum from rapamycin-treated VPA-induced cells. (B) Graph of viral titers untreated (black bars) or treated with rapamycin (gray bars) for each indicated inducting agent. Representative experiment, n = 2. (C) Inhibition of cell-to-cell virus transmission from spontaneously lytic BCBL-1 was assessed by culture of BCBL-1 in the presence of 12 nM rapamycin for 2 days. Cells were then harvested, placed in fresh media without rapamycin and co-cultured with HeLa cells for 24 h. BCBL-1 cells were removed, HeLa cells washed x 2 to remove non-adherent BCBL-1 cells, and cultured an additional 24 h before harvest. Adherent HeLa cells were trypsinized, fixed and stained for intranuclear LANA dots. LANA+ HeLa cells were analyzed by MIFC. Graph shows percentage (mean ± s.d.) of infected cells from triplicate cultures for HeLa cells co-cultured with either rapamycin-treated (gray) or DMSO-treated (black) BCBL-1. ** p<0.01.
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pone-0014535-g005: Virion production is significantly decreased by rapamycin treatment.BCBL-1 +/− rapamycin 12 nM were induced with CoCl2, TPA, or VPA. Five days post-induction, virus was concentrated from supernatants. HeLa cells were then infected with concentrated virus in the presence of polybrene. Viral titers were determined by staining HeLa cells for punctate intranuclear LANA dots and analyzing via multispectral imaging flow cytometry. (A) Images show representative nuclear staining with DRAQ5 (Nuc.), LANA antibody stain, and the overlay (Comb.). Upper panel shows three representative HeLa cells infected with inoculum from untreated VPA-induced BCBL-1. Lower panel shows representative HeLas infected with inoculum from rapamycin-treated VPA-induced cells. (B) Graph of viral titers untreated (black bars) or treated with rapamycin (gray bars) for each indicated inducting agent. Representative experiment, n = 2. (C) Inhibition of cell-to-cell virus transmission from spontaneously lytic BCBL-1 was assessed by culture of BCBL-1 in the presence of 12 nM rapamycin for 2 days. Cells were then harvested, placed in fresh media without rapamycin and co-cultured with HeLa cells for 24 h. BCBL-1 cells were removed, HeLa cells washed x 2 to remove non-adherent BCBL-1 cells, and cultured an additional 24 h before harvest. Adherent HeLa cells were trypsinized, fixed and stained for intranuclear LANA dots. LANA+ HeLa cells were analyzed by MIFC. Graph shows percentage (mean ± s.d.) of infected cells from triplicate cultures for HeLa cells co-cultured with either rapamycin-treated (gray) or DMSO-treated (black) BCBL-1. ** p<0.01.

Mentions: While RTA is necessary and sufficient for initiating viral lytic replication, it is only the initial measure of the productive phase of infection. Clinically, the most important read-out is the production of virions. To assess this parameter, we treated BCBL-1 cells with rapamycin, induced with VPA, TPA, or CoCl2 and then measured viral titers five days later. As we have shown previously in KSHV-infected cells, the number of punctate aggregates of virally-encoded latency-associated nuclear antigen (LANA), or “LANA dots”, correspond to the number of KSHV episomes present in the nucleus[38]. Thus, following de novo infection of KSHV-susceptible cells, the resulting LANA dots reflect the titer in the inoculum. Briefly, following each treatment we harvested media pre-cleared of cellular debris, collected the virus by centrifugation, and then incubated serial dilutions of the resuspended viral pellet with HeLa cells in the presence of polybrene. We measured titers of infectious virus by staining the KSHV-exposed HeLa cells for LANA and analyzing cells using multispectral imaging flow cytometry (MIFC) to determine the number of punctate LANA dots per cell. As evident in Figure 5A, rapamycin treatment decreased the amount of virus produced from BCBL-1 cells treated with VPA, TPA, or CoCl2 by 9.3, 9.7, and 7.7 fold, respectively.


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)

Virion production is significantly decreased by rapamycin treatment.BCBL-1 +/− rapamycin 12 nM were induced with CoCl2, TPA, or VPA. Five days post-induction, virus was concentrated from supernatants. HeLa cells were then infected with concentrated virus in the presence of polybrene. Viral titers were determined by staining HeLa cells for punctate intranuclear LANA dots and analyzing via multispectral imaging flow cytometry. (A) Images show representative nuclear staining with DRAQ5 (Nuc.), LANA antibody stain, and the overlay (Comb.). Upper panel shows three representative HeLa cells infected with inoculum from untreated VPA-induced BCBL-1. Lower panel shows representative HeLas infected with inoculum from rapamycin-treated VPA-induced cells. (B) Graph of viral titers untreated (black bars) or treated with rapamycin (gray bars) for each indicated inducting agent. Representative experiment, n = 2. (C) Inhibition of cell-to-cell virus transmission from spontaneously lytic BCBL-1 was assessed by culture of BCBL-1 in the presence of 12 nM rapamycin for 2 days. Cells were then harvested, placed in fresh media without rapamycin and co-cultured with HeLa cells for 24 h. BCBL-1 cells were removed, HeLa cells washed x 2 to remove non-adherent BCBL-1 cells, and cultured an additional 24 h before harvest. Adherent HeLa cells were trypsinized, fixed and stained for intranuclear LANA dots. LANA+ HeLa cells were analyzed by MIFC. Graph shows percentage (mean ± s.d.) of infected cells from triplicate cultures for HeLa cells co-cultured with either rapamycin-treated (gray) or DMSO-treated (black) BCBL-1. ** p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021514&req=5

pone-0014535-g005: Virion production is significantly decreased by rapamycin treatment.BCBL-1 +/− rapamycin 12 nM were induced with CoCl2, TPA, or VPA. Five days post-induction, virus was concentrated from supernatants. HeLa cells were then infected with concentrated virus in the presence of polybrene. Viral titers were determined by staining HeLa cells for punctate intranuclear LANA dots and analyzing via multispectral imaging flow cytometry. (A) Images show representative nuclear staining with DRAQ5 (Nuc.), LANA antibody stain, and the overlay (Comb.). Upper panel shows three representative HeLa cells infected with inoculum from untreated VPA-induced BCBL-1. Lower panel shows representative HeLas infected with inoculum from rapamycin-treated VPA-induced cells. (B) Graph of viral titers untreated (black bars) or treated with rapamycin (gray bars) for each indicated inducting agent. Representative experiment, n = 2. (C) Inhibition of cell-to-cell virus transmission from spontaneously lytic BCBL-1 was assessed by culture of BCBL-1 in the presence of 12 nM rapamycin for 2 days. Cells were then harvested, placed in fresh media without rapamycin and co-cultured with HeLa cells for 24 h. BCBL-1 cells were removed, HeLa cells washed x 2 to remove non-adherent BCBL-1 cells, and cultured an additional 24 h before harvest. Adherent HeLa cells were trypsinized, fixed and stained for intranuclear LANA dots. LANA+ HeLa cells were analyzed by MIFC. Graph shows percentage (mean ± s.d.) of infected cells from triplicate cultures for HeLa cells co-cultured with either rapamycin-treated (gray) or DMSO-treated (black) BCBL-1. ** p<0.01.
Mentions: While RTA is necessary and sufficient for initiating viral lytic replication, it is only the initial measure of the productive phase of infection. Clinically, the most important read-out is the production of virions. To assess this parameter, we treated BCBL-1 cells with rapamycin, induced with VPA, TPA, or CoCl2 and then measured viral titers five days later. As we have shown previously in KSHV-infected cells, the number of punctate aggregates of virally-encoded latency-associated nuclear antigen (LANA), or “LANA dots”, correspond to the number of KSHV episomes present in the nucleus[38]. Thus, following de novo infection of KSHV-susceptible cells, the resulting LANA dots reflect the titer in the inoculum. Briefly, following each treatment we harvested media pre-cleared of cellular debris, collected the virus by centrifugation, and then incubated serial dilutions of the resuspended viral pellet with HeLa cells in the presence of polybrene. We measured titers of infectious virus by staining the KSHV-exposed HeLa cells for LANA and analyzing cells using multispectral imaging flow cytometry (MIFC) to determine the number of punctate LANA dots per cell. As evident in Figure 5A, rapamycin treatment decreased the amount of virus produced from BCBL-1 cells treated with VPA, TPA, or CoCl2 by 9.3, 9.7, and 7.7 fold, respectively.

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