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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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HLA-DRα is down-regulated in LANA-expressing B lymphoma cells.(A) The mRNA level of HLA-DRα was reduced in LANA-expressing BJAB cells. The total RNA of BJAB cells with LANA-RFP or vector RFP stable expression were subjected to microarray assay. Representative data and average ratio of HLA-DRα, CIITA and CREB from LANA-RFP and RFP microarray assay are shown. (B) Northern analysis of HLA-DRα transcript. The total RNA of BJAB and DG75 cells individually transduced with lentiviruses carrying LANA (YLF) or vector (GFP) alone was subjected to northern blot as described in material and method. The relative density of HLA-DRα transcript is verified by quantitative PCR. The efficiency of lentivirus transduction is shown at the right panels. (C) LANA inhibits the expression of HLA-DRα. Ten million of DG75 cells were electroporated with pA3M-LANA (5, 10, 15 µg) or Vector pA3M. At 48-hr post-transfection, the cell lysate were separated by SDS-PAGE and analyzed by Western blot using antibodies HLA-DRα or myc(9E10). Detection of GAPDH was used as an internal control. (D) The surface expression of HLA-DR proteins on DG75 cells transfected with LANA-myc or vector was determined by FACS using PE-labeled mAb against HLA-DR.
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ppat-1003751-g001: HLA-DRα is down-regulated in LANA-expressing B lymphoma cells.(A) The mRNA level of HLA-DRα was reduced in LANA-expressing BJAB cells. The total RNA of BJAB cells with LANA-RFP or vector RFP stable expression were subjected to microarray assay. Representative data and average ratio of HLA-DRα, CIITA and CREB from LANA-RFP and RFP microarray assay are shown. (B) Northern analysis of HLA-DRα transcript. The total RNA of BJAB and DG75 cells individually transduced with lentiviruses carrying LANA (YLF) or vector (GFP) alone was subjected to northern blot as described in material and method. The relative density of HLA-DRα transcript is verified by quantitative PCR. The efficiency of lentivirus transduction is shown at the right panels. (C) LANA inhibits the expression of HLA-DRα. Ten million of DG75 cells were electroporated with pA3M-LANA (5, 10, 15 µg) or Vector pA3M. At 48-hr post-transfection, the cell lysate were separated by SDS-PAGE and analyzed by Western blot using antibodies HLA-DRα or myc(9E10). Detection of GAPDH was used as an internal control. (D) The surface expression of HLA-DR proteins on DG75 cells transfected with LANA-myc or vector was determined by FACS using PE-labeled mAb against HLA-DR.

Mentions: LANA has been shown to widely interact with several cellular transcription factors. To analyze the overall outcome of these interactions at the level of the cellular gene transcriptome related to immune regulation, the B lymphoma BJAB cells with LANA-RFP stably expressed or RFP vector alone were established and immunology/hematology-related gene mRNA expression profiles were determined by microarray. Microarray analysis revealed that HLA-DRα (a MHC II molecule), CIITA and CREB transcripts were consistently down-regulated more than 18-fold upon LANA expression (Figure 1A). The observed down-regulation of HLA-DRα upon LANA expression was verified by northern blot analysis in both BJAB and DG75 cells transduced with LANA-expressing Lentivirus (YLF) when compared with vector alone control (GFP) at levels ranging from 4–8 fold (Figure 1B). This was further confirmed by the fact that LANA induces a dose-dependent decrease in HLA-DRα expression in DG75 cells with transient transfection up to about 5-fold as expected when compared to the northern blot above (Figure 1C). To determine if HLA-DR presentation was affected by LANA, the levels of HLA-DR at the cell surface in the presence or absence of LANA was determined by flow cytometric analysis. The results showed that LANA dramatically reduced HLA-DRα presentation at the cell surface as seen by the shift in fluorescence peak for LANA-expressing cells (Figure 1D).


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)

HLA-DRα is down-regulated in LANA-expressing B lymphoma cells.(A) The mRNA level of HLA-DRα was reduced in LANA-expressing BJAB cells. The total RNA of BJAB cells with LANA-RFP or vector RFP stable expression were subjected to microarray assay. Representative data and average ratio of HLA-DRα, CIITA and CREB from LANA-RFP and RFP microarray assay are shown. (B) Northern analysis of HLA-DRα transcript. The total RNA of BJAB and DG75 cells individually transduced with lentiviruses carrying LANA (YLF) or vector (GFP) alone was subjected to northern blot as described in material and method. The relative density of HLA-DRα transcript is verified by quantitative PCR. The efficiency of lentivirus transduction is shown at the right panels. (C) LANA inhibits the expression of HLA-DRα. Ten million of DG75 cells were electroporated with pA3M-LANA (5, 10, 15 µg) or Vector pA3M. At 48-hr post-transfection, the cell lysate were separated by SDS-PAGE and analyzed by Western blot using antibodies HLA-DRα or myc(9E10). Detection of GAPDH was used as an internal control. (D) The surface expression of HLA-DR proteins on DG75 cells transfected with LANA-myc or vector was determined by FACS using PE-labeled mAb against HLA-DR.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814934&req=5

ppat-1003751-g001: HLA-DRα is down-regulated in LANA-expressing B lymphoma cells.(A) The mRNA level of HLA-DRα was reduced in LANA-expressing BJAB cells. The total RNA of BJAB cells with LANA-RFP or vector RFP stable expression were subjected to microarray assay. Representative data and average ratio of HLA-DRα, CIITA and CREB from LANA-RFP and RFP microarray assay are shown. (B) Northern analysis of HLA-DRα transcript. The total RNA of BJAB and DG75 cells individually transduced with lentiviruses carrying LANA (YLF) or vector (GFP) alone was subjected to northern blot as described in material and method. The relative density of HLA-DRα transcript is verified by quantitative PCR. The efficiency of lentivirus transduction is shown at the right panels. (C) LANA inhibits the expression of HLA-DRα. Ten million of DG75 cells were electroporated with pA3M-LANA (5, 10, 15 µg) or Vector pA3M. At 48-hr post-transfection, the cell lysate were separated by SDS-PAGE and analyzed by Western blot using antibodies HLA-DRα or myc(9E10). Detection of GAPDH was used as an internal control. (D) The surface expression of HLA-DR proteins on DG75 cells transfected with LANA-myc or vector was determined by FACS using PE-labeled mAb against HLA-DR.
Mentions: LANA has been shown to widely interact with several cellular transcription factors. To analyze the overall outcome of these interactions at the level of the cellular gene transcriptome related to immune regulation, the B lymphoma BJAB cells with LANA-RFP stably expressed or RFP vector alone were established and immunology/hematology-related gene mRNA expression profiles were determined by microarray. Microarray analysis revealed that HLA-DRα (a MHC II molecule), CIITA and CREB transcripts were consistently down-regulated more than 18-fold upon LANA expression (Figure 1A). The observed down-regulation of HLA-DRα upon LANA expression was verified by northern blot analysis in both BJAB and DG75 cells transduced with LANA-expressing Lentivirus (YLF) when compared with vector alone control (GFP) at levels ranging from 4–8 fold (Figure 1B). This was further confirmed by the fact that LANA induces a dose-dependent decrease in HLA-DRα expression in DG75 cells with transient transfection up to about 5-fold as expected when compared to the northern blot above (Figure 1C). To determine if HLA-DR presentation was affected by LANA, the levels of HLA-DR at the cell surface in the presence or absence of LANA was determined by flow cytometric analysis. The results showed that LANA dramatically reduced HLA-DRα presentation at the cell surface as seen by the shift in fluorescence peak for LANA-expressing cells (Figure 1D).

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