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Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

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Related in: MedlinePlus

MEF-2 inhibition reduces HTLV-1 LTR transactivation, Tax expression, and viral replication. (A) Transient transfection of Jurkat cells with pU3R-luc (HTLV-1 LTR luciferase reporter construct) as well as plasmids that express Tax, MEF-2A, HDAC9 and MEF-2A shRNA, was done as described in Methods. Before co-transfecting two or more plasmids, each of these plasmids was transfected alone to establish the background levels of luciferase activity. Cells were collected 24 hr post-transfection, lysed and assayed using the dual luciferase assay system. Firefly luciferase activity was normalized with that of Renilla luciferase expressed from phRL/CMV. Each bar represents the average of triplicate samples. Significance among groups was derived by student’s t-test to determine the p-value. (*p < 0.05). (B) MT-2 cells were transfected with either scrambled or shMEF-2 plasmid. Western blot analysis was performed at 24 hr and 48 hr to determine protein levels of MEF-2, Tax, and beta-actin. Data represent one of two separate experiments. (C) To analyze effects of shMEF-2A on virus production, transfected MT-2 cells were washed at 48 hr and incubated in fresh medium for another 24–36 hr. Thereafter, supernatants were assessed for HTLV-1 core protein levels (pg/ml) by the p19-specific ELISA (ZeptoMetrix, Buffalo, NY). (D) MT-2 cells were transfected either with a mock plasmid or MITR/HDAC9 plasmids followed by cell collection at every 24 hr over a 72 hr period. Real-time PCR analyses were performed to determine relative mRNA levels of Tax and p19. Data is representative of at least three independent experiments.
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Fig1: MEF-2 inhibition reduces HTLV-1 LTR transactivation, Tax expression, and viral replication. (A) Transient transfection of Jurkat cells with pU3R-luc (HTLV-1 LTR luciferase reporter construct) as well as plasmids that express Tax, MEF-2A, HDAC9 and MEF-2A shRNA, was done as described in Methods. Before co-transfecting two or more plasmids, each of these plasmids was transfected alone to establish the background levels of luciferase activity. Cells were collected 24 hr post-transfection, lysed and assayed using the dual luciferase assay system. Firefly luciferase activity was normalized with that of Renilla luciferase expressed from phRL/CMV. Each bar represents the average of triplicate samples. Significance among groups was derived by student’s t-test to determine the p-value. (*p < 0.05). (B) MT-2 cells were transfected with either scrambled or shMEF-2 plasmid. Western blot analysis was performed at 24 hr and 48 hr to determine protein levels of MEF-2, Tax, and beta-actin. Data represent one of two separate experiments. (C) To analyze effects of shMEF-2A on virus production, transfected MT-2 cells were washed at 48 hr and incubated in fresh medium for another 24–36 hr. Thereafter, supernatants were assessed for HTLV-1 core protein levels (pg/ml) by the p19-specific ELISA (ZeptoMetrix, Buffalo, NY). (D) MT-2 cells were transfected either with a mock plasmid or MITR/HDAC9 plasmids followed by cell collection at every 24 hr over a 72 hr period. Real-time PCR analyses were performed to determine relative mRNA levels of Tax and p19. Data is representative of at least three independent experiments.

Mentions: In order to understand effects of MEF-2 on transcriptional activation of HTLV-1 LTR, we transfected Jurkat cells with pU3R-luc (HTLV-1 5′ LTR luciferase reporter vector), and plasmids expressing Tax (pCMV-Tax), MEF-2A (p3X-Luc-MEF-2), and HDAC9 (pHDAC9). Also, the pLKO.1-puro shMEF-2A plasmid was used to knock down MEF-2A expression. As expected, LTR activity significantly increased in the presence of Tax (Figure 1A) Overexpression of MEF-2A, in the absence of Tax did not have any impact on LTR activation but showed slight enhancement on Tax activity with a p-value of 0.1. On the other hand, inhibition of MEF-2A expression by shRNA or its activity by HDAC9 demonstrated significant reduction in Tax-mediated LTR activation (Figure 1A), suggesting that Tax partners with cellular MEF-2 during the transactivation process. Each plasmid was titrated at various concentrations and was used at an optimal dose. Transfection efficiency was measured using a pMX-GFP plasmid (Lonza) and ranged from 58-64% in triplicate samples (Additional file 1: Figure S1A). Besides Tax plasmid no other plasmid has any direct effect on LTR activation providing internal control to the assay and avoiding the possibility of a general suppression with shMEF-2A and/or pHDAC9. For other controls, scrambled shRNA was used, MEF-2 inhibition was confirmed by end-point RT-PCR (Additional file 1: Figure S1B), and LDH cytotoxicity assay was performed to measure extracellular LDH in transfection media to confirm comparable viability of transfected cells among experimental variables (Additional file 1: Figure S1C).Figure 1


Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB.

Jain P, Lavorgna A, Sehgal M, Gao L, Ginwala R, Sagar D, Harhaj EW, Khan ZK - Retrovirology (2015)

MEF-2 inhibition reduces HTLV-1 LTR transactivation, Tax expression, and viral replication. (A) Transient transfection of Jurkat cells with pU3R-luc (HTLV-1 LTR luciferase reporter construct) as well as plasmids that express Tax, MEF-2A, HDAC9 and MEF-2A shRNA, was done as described in Methods. Before co-transfecting two or more plasmids, each of these plasmids was transfected alone to establish the background levels of luciferase activity. Cells were collected 24 hr post-transfection, lysed and assayed using the dual luciferase assay system. Firefly luciferase activity was normalized with that of Renilla luciferase expressed from phRL/CMV. Each bar represents the average of triplicate samples. Significance among groups was derived by student’s t-test to determine the p-value. (*p < 0.05). (B) MT-2 cells were transfected with either scrambled or shMEF-2 plasmid. Western blot analysis was performed at 24 hr and 48 hr to determine protein levels of MEF-2, Tax, and beta-actin. Data represent one of two separate experiments. (C) To analyze effects of shMEF-2A on virus production, transfected MT-2 cells were washed at 48 hr and incubated in fresh medium for another 24–36 hr. Thereafter, supernatants were assessed for HTLV-1 core protein levels (pg/ml) by the p19-specific ELISA (ZeptoMetrix, Buffalo, NY). (D) MT-2 cells were transfected either with a mock plasmid or MITR/HDAC9 plasmids followed by cell collection at every 24 hr over a 72 hr period. Real-time PCR analyses were performed to determine relative mRNA levels of Tax and p19. Data is representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374383&req=5

Fig1: MEF-2 inhibition reduces HTLV-1 LTR transactivation, Tax expression, and viral replication. (A) Transient transfection of Jurkat cells with pU3R-luc (HTLV-1 LTR luciferase reporter construct) as well as plasmids that express Tax, MEF-2A, HDAC9 and MEF-2A shRNA, was done as described in Methods. Before co-transfecting two or more plasmids, each of these plasmids was transfected alone to establish the background levels of luciferase activity. Cells were collected 24 hr post-transfection, lysed and assayed using the dual luciferase assay system. Firefly luciferase activity was normalized with that of Renilla luciferase expressed from phRL/CMV. Each bar represents the average of triplicate samples. Significance among groups was derived by student’s t-test to determine the p-value. (*p < 0.05). (B) MT-2 cells were transfected with either scrambled or shMEF-2 plasmid. Western blot analysis was performed at 24 hr and 48 hr to determine protein levels of MEF-2, Tax, and beta-actin. Data represent one of two separate experiments. (C) To analyze effects of shMEF-2A on virus production, transfected MT-2 cells were washed at 48 hr and incubated in fresh medium for another 24–36 hr. Thereafter, supernatants were assessed for HTLV-1 core protein levels (pg/ml) by the p19-specific ELISA (ZeptoMetrix, Buffalo, NY). (D) MT-2 cells were transfected either with a mock plasmid or MITR/HDAC9 plasmids followed by cell collection at every 24 hr over a 72 hr period. Real-time PCR analyses were performed to determine relative mRNA levels of Tax and p19. Data is representative of at least three independent experiments.
Mentions: In order to understand effects of MEF-2 on transcriptional activation of HTLV-1 LTR, we transfected Jurkat cells with pU3R-luc (HTLV-1 5′ LTR luciferase reporter vector), and plasmids expressing Tax (pCMV-Tax), MEF-2A (p3X-Luc-MEF-2), and HDAC9 (pHDAC9). Also, the pLKO.1-puro shMEF-2A plasmid was used to knock down MEF-2A expression. As expected, LTR activity significantly increased in the presence of Tax (Figure 1A) Overexpression of MEF-2A, in the absence of Tax did not have any impact on LTR activation but showed slight enhancement on Tax activity with a p-value of 0.1. On the other hand, inhibition of MEF-2A expression by shRNA or its activity by HDAC9 demonstrated significant reduction in Tax-mediated LTR activation (Figure 1A), suggesting that Tax partners with cellular MEF-2 during the transactivation process. Each plasmid was titrated at various concentrations and was used at an optimal dose. Transfection efficiency was measured using a pMX-GFP plasmid (Lonza) and ranged from 58-64% in triplicate samples (Additional file 1: Figure S1A). Besides Tax plasmid no other plasmid has any direct effect on LTR activation providing internal control to the assay and avoiding the possibility of a general suppression with shMEF-2A and/or pHDAC9. For other controls, scrambled shRNA was used, MEF-2 inhibition was confirmed by end-point RT-PCR (Additional file 1: Figure S1B), and LDH cytotoxicity assay was performed to measure extracellular LDH in transfection media to confirm comparable viability of transfected cells among experimental variables (Additional file 1: Figure S1C).Figure 1

Bottom Line: Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase).MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.

Results: Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.

Conclusions: We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

Show MeSH
Related in: MedlinePlus