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IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation.

Cai Q, Banerjee S, Cervini A, Lu J, Hislop AD, Dzeng R, Robertson ES - PLoS Pathog. (2013)

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear.Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored.Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters).

View Article: PubMed Central - PubMed

Affiliation: MOE&MOH Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China.

ABSTRACT
Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHC II expression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHC II expression and presentation by inhibiting the transcription of MHC II transactivator (CIITA) promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters). This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHC II presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.

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LANA expression represses CIITA expression in B lymphoma cells.(A) The level of CIITA expression was reduced by LANA in a dose-dependent manner. Ten million of DG75 or Ramos cells were transfected with a different dose of DNA construct expressing LANA. At 24 hr post-transfection, cells were harvested and lysed for immunoblotting assays as indicated in the figure. GAPDH blot was used as loading control. The relative density (RD) of CIITA proteins was quantified and shown in the figure. (B) Schematic representation of CIITA promoter reporters (pIII-Luc and pIV-Luc) with luciferase. (C) DG75 and Ramos cells were individually co-transfected CIITA promoter (pIII and pIV) driving luciferase reporter plasmid with either pA3M-LANA or pA3M vector. At 24-hrs post-transfection, cells were harvested and subjected to reporter assay. The results were presented by the RLU (relative luciferase unit) fold compared to pGL3-basic with vector alone. Data is presented as means±SD of three independent experiments. The expression of LANA were detected by immunoblotting and shown at the bottom panels. (D) Quantitative real-time PCR analysis of MHCII in DG75 cells transfected with different dose LANA as in panel A.
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ppat-1003751-g003: LANA expression represses CIITA expression in B lymphoma cells.(A) The level of CIITA expression was reduced by LANA in a dose-dependent manner. Ten million of DG75 or Ramos cells were transfected with a different dose of DNA construct expressing LANA. At 24 hr post-transfection, cells were harvested and lysed for immunoblotting assays as indicated in the figure. GAPDH blot was used as loading control. The relative density (RD) of CIITA proteins was quantified and shown in the figure. (B) Schematic representation of CIITA promoter reporters (pIII-Luc and pIV-Luc) with luciferase. (C) DG75 and Ramos cells were individually co-transfected CIITA promoter (pIII and pIV) driving luciferase reporter plasmid with either pA3M-LANA or pA3M vector. At 24-hrs post-transfection, cells were harvested and subjected to reporter assay. The results were presented by the RLU (relative luciferase unit) fold compared to pGL3-basic with vector alone. Data is presented as means±SD of three independent experiments. The expression of LANA were detected by immunoblotting and shown at the bottom panels. (D) Quantitative real-time PCR analysis of MHCII in DG75 cells transfected with different dose LANA as in panel A.

Mentions: To confirm if LANA down-regulates CIITA in a dose-dependent manner, we performed luciferase reporter assays by using CIITA pIII or pIV promoter-driven luciferase gene expression, as well as detected endogenous CIITA protein levels by immunoblotting in both DG75 and Ramos B lymphoma cells with an increasing dose of LANA. The results showed that the reduced CIITA protein levels of 5–9 fold were seen with increased LANA expression in the two B-cell line Ramos and DG75 (Figure 3A). Consistently, similar results showing transcriptional inhibition of both pIII and pIV promoters by LANA from the reporter assays in Ramos and DG75 (Figure 3B and C), strongly indicate that down-regulation of CIITA transcripts were tightly associated with LANA expression during KSHV infection. Importantly, the rapid inhibition of MHC II expression during early infection could be critical for KSHV to establish long-term latent infection (which at least is partially contributed by LANA expression, as shown in Figure 3D), we also observed there was a dramatic dose-dependent inhibition of MHC II expression in the presence of LANA alone.


IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation.

Cai Q, Banerjee S, Cervini A, Lu J, Hislop AD, Dzeng R, Robertson ES - PLoS Pathog. (2013)

LANA expression represses CIITA expression in B lymphoma cells.(A) The level of CIITA expression was reduced by LANA in a dose-dependent manner. Ten million of DG75 or Ramos cells were transfected with a different dose of DNA construct expressing LANA. At 24 hr post-transfection, cells were harvested and lysed for immunoblotting assays as indicated in the figure. GAPDH blot was used as loading control. The relative density (RD) of CIITA proteins was quantified and shown in the figure. (B) Schematic representation of CIITA promoter reporters (pIII-Luc and pIV-Luc) with luciferase. (C) DG75 and Ramos cells were individually co-transfected CIITA promoter (pIII and pIV) driving luciferase reporter plasmid with either pA3M-LANA or pA3M vector. At 24-hrs post-transfection, cells were harvested and subjected to reporter assay. The results were presented by the RLU (relative luciferase unit) fold compared to pGL3-basic with vector alone. Data is presented as means±SD of three independent experiments. The expression of LANA were detected by immunoblotting and shown at the bottom panels. (D) Quantitative real-time PCR analysis of MHCII in DG75 cells transfected with different dose LANA as in panel A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814934&req=5

ppat-1003751-g003: LANA expression represses CIITA expression in B lymphoma cells.(A) The level of CIITA expression was reduced by LANA in a dose-dependent manner. Ten million of DG75 or Ramos cells were transfected with a different dose of DNA construct expressing LANA. At 24 hr post-transfection, cells were harvested and lysed for immunoblotting assays as indicated in the figure. GAPDH blot was used as loading control. The relative density (RD) of CIITA proteins was quantified and shown in the figure. (B) Schematic representation of CIITA promoter reporters (pIII-Luc and pIV-Luc) with luciferase. (C) DG75 and Ramos cells were individually co-transfected CIITA promoter (pIII and pIV) driving luciferase reporter plasmid with either pA3M-LANA or pA3M vector. At 24-hrs post-transfection, cells were harvested and subjected to reporter assay. The results were presented by the RLU (relative luciferase unit) fold compared to pGL3-basic with vector alone. Data is presented as means±SD of three independent experiments. The expression of LANA were detected by immunoblotting and shown at the bottom panels. (D) Quantitative real-time PCR analysis of MHCII in DG75 cells transfected with different dose LANA as in panel A.
Mentions: To confirm if LANA down-regulates CIITA in a dose-dependent manner, we performed luciferase reporter assays by using CIITA pIII or pIV promoter-driven luciferase gene expression, as well as detected endogenous CIITA protein levels by immunoblotting in both DG75 and Ramos B lymphoma cells with an increasing dose of LANA. The results showed that the reduced CIITA protein levels of 5–9 fold were seen with increased LANA expression in the two B-cell line Ramos and DG75 (Figure 3A). Consistently, similar results showing transcriptional inhibition of both pIII and pIV promoters by LANA from the reporter assays in Ramos and DG75 (Figure 3B and C), strongly indicate that down-regulation of CIITA transcripts were tightly associated with LANA expression during KSHV infection. Importantly, the rapid inhibition of MHC II expression during early infection could be critical for KSHV to establish long-term latent infection (which at least is partially contributed by LANA expression, as shown in Figure 3D), we also observed there was a dramatic dose-dependent inhibition of MHC II expression in the presence of LANA alone.

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear.Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored.Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters).

View Article: PubMed Central - PubMed

Affiliation: MOE&MOH Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China.

ABSTRACT
Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHC II expression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHC II expression and presentation by inhibiting the transcription of MHC II transactivator (CIITA) promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters). This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHC II presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.

Show MeSH
Related in: MedlinePlus