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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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IRF-4 binds with LANA through its DNA-binding domain in vitro.(A) IRF-4 binds to C-terminal domain of LANA in vitro. The 35S-radiolabeled in vitro-translated proteins of LANA truncated mutants were pre-cleared with GST bead, followed by incubation with GST or GST-IRF-4 beads. The bound protein mixtures were resolved by appropriate SDS-PAGE, and protein species detected by autoradiography. 5% of in vitro translated protein is used as input. The quantification of relative amount of bound proteins (RBU) is shown at the bottom. (B) The 35S-radiolabeled in vitro-translated full length IRF-4 was pulled down by truncated mutants of LANA fusion with GST (GST-LANA N1–340 and C945–1162) in the presence or absence of DNase I (20 U/ml) treatment. Coomassie blue staining of purified GST-LANA is shown at the bottom panels. (C) LANA binds to N-terminal domain of IRF-4 in vitro. The 35S-radiolabeled in vitro-translated proteins IRF-4 with FLAG tag (1–135, 1–271, 145–451 and 246–451) was pulled down by GST or GST-LANA C. Schematics illustrate different structural domains of IRF-4 with LANA-binding ability.
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ppat-1003751-g006: IRF-4 binds with LANA through its DNA-binding domain in vitro.(A) IRF-4 binds to C-terminal domain of LANA in vitro. The 35S-radiolabeled in vitro-translated proteins of LANA truncated mutants were pre-cleared with GST bead, followed by incubation with GST or GST-IRF-4 beads. The bound protein mixtures were resolved by appropriate SDS-PAGE, and protein species detected by autoradiography. 5% of in vitro translated protein is used as input. The quantification of relative amount of bound proteins (RBU) is shown at the bottom. (B) The 35S-radiolabeled in vitro-translated full length IRF-4 was pulled down by truncated mutants of LANA fusion with GST (GST-LANA N1–340 and C945–1162) in the presence or absence of DNase I (20 U/ml) treatment. Coomassie blue staining of purified GST-LANA is shown at the bottom panels. (C) LANA binds to N-terminal domain of IRF-4 in vitro. The 35S-radiolabeled in vitro-translated proteins IRF-4 with FLAG tag (1–135, 1–271, 145–451 and 246–451) was pulled down by GST or GST-LANA C. Schematics illustrate different structural domains of IRF-4 with LANA-binding ability.

Mentions: To determine if IRF-4 directly interacts with LANA, we performed GST pull-down assays by using a series of in vitro-translated truncated mutants of LANA plus the full length molecule incubated with bacterially expressed GST-IRF-4 or GST alone protein. The results showed that the C-terminal domain (945–1162) of LANA strongly interacted with IRF-4 (Figure 6A). Similarly, using in vitro-translated IRF-4 co-incubated with bacterially expressed GST-LANA N (1–340) and C (945–1162), the results of GST pull-down assays confirmed that the C-terminus of LANA directly binds with IRF-4 (Figure 6B). To define which domain of IRF-4 is responsible for interacting with LANA, we generated four truncated mutants of IRF-4 with FLAG tag residues (1–271, 145–451, 246–451 and 1–135) and performed similar GST pull-down assays again by using GST-LANA C incubated with in vitro-translated truncated mutants of IRF-4. The results showed that IRF-4 bound with LANA through its DNA-binding domain (residues 1–135) at the N-terminus (Figure 6C). The results which showed that in-vitro translated IRF-4 treated with DNase I still bound to LANA (Figure 6B), excluded the possibility that IRF-4 and LANA interaction was dependent on the DNA sequence. To confirm that these two molecules do associate in cells, we performed co-immunoprecipitation assays to determine if LANA forms a complex with IRF-4 in KSHV negative (DG75) and positive (BC3) B-lymphoma cell lines. The results of the co-IP assays from DG75 cells with exogenous LANA and IRF-4 co-expression, showed a complex of IRF-4 with LANA (Figure 7A, lane 6). Consistent with these results, the co-IP studies from BC3 cells showed that endogenous IRF-4 was dramatically immunoprecipitated with anti-LANA antibody, but not using a control mouse serum (Figure 7B, top and left panels). The reverse immunoprecipitation using anti-IRF-4 antibody further confirmed that LANA does form a complex with IRF-4 in KSHV infected cells (Figure 7B, top and right panels). This supports the data above that these two proteins can associate in the same molecular complex.


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)

IRF-4 binds with LANA through its DNA-binding domain in vitro.(A) IRF-4 binds to C-terminal domain of LANA in vitro. The 35S-radiolabeled in vitro-translated proteins of LANA truncated mutants were pre-cleared with GST bead, followed by incubation with GST or GST-IRF-4 beads. The bound protein mixtures were resolved by appropriate SDS-PAGE, and protein species detected by autoradiography. 5% of in vitro translated protein is used as input. The quantification of relative amount of bound proteins (RBU) is shown at the bottom. (B) The 35S-radiolabeled in vitro-translated full length IRF-4 was pulled down by truncated mutants of LANA fusion with GST (GST-LANA N1–340 and C945–1162) in the presence or absence of DNase I (20 U/ml) treatment. Coomassie blue staining of purified GST-LANA is shown at the bottom panels. (C) LANA binds to N-terminal domain of IRF-4 in vitro. The 35S-radiolabeled in vitro-translated proteins IRF-4 with FLAG tag (1–135, 1–271, 145–451 and 246–451) was pulled down by GST or GST-LANA C. Schematics illustrate different structural domains of IRF-4 with LANA-binding ability.
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Related In: Results  -  Collection

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

ppat-1003751-g006: IRF-4 binds with LANA through its DNA-binding domain in vitro.(A) IRF-4 binds to C-terminal domain of LANA in vitro. The 35S-radiolabeled in vitro-translated proteins of LANA truncated mutants were pre-cleared with GST bead, followed by incubation with GST or GST-IRF-4 beads. The bound protein mixtures were resolved by appropriate SDS-PAGE, and protein species detected by autoradiography. 5% of in vitro translated protein is used as input. The quantification of relative amount of bound proteins (RBU) is shown at the bottom. (B) The 35S-radiolabeled in vitro-translated full length IRF-4 was pulled down by truncated mutants of LANA fusion with GST (GST-LANA N1–340 and C945–1162) in the presence or absence of DNase I (20 U/ml) treatment. Coomassie blue staining of purified GST-LANA is shown at the bottom panels. (C) LANA binds to N-terminal domain of IRF-4 in vitro. The 35S-radiolabeled in vitro-translated proteins IRF-4 with FLAG tag (1–135, 1–271, 145–451 and 246–451) was pulled down by GST or GST-LANA C. Schematics illustrate different structural domains of IRF-4 with LANA-binding ability.
Mentions: To determine if IRF-4 directly interacts with LANA, we performed GST pull-down assays by using a series of in vitro-translated truncated mutants of LANA plus the full length molecule incubated with bacterially expressed GST-IRF-4 or GST alone protein. The results showed that the C-terminal domain (945–1162) of LANA strongly interacted with IRF-4 (Figure 6A). Similarly, using in vitro-translated IRF-4 co-incubated with bacterially expressed GST-LANA N (1–340) and C (945–1162), the results of GST pull-down assays confirmed that the C-terminus of LANA directly binds with IRF-4 (Figure 6B). To define which domain of IRF-4 is responsible for interacting with LANA, we generated four truncated mutants of IRF-4 with FLAG tag residues (1–271, 145–451, 246–451 and 1–135) and performed similar GST pull-down assays again by using GST-LANA C incubated with in vitro-translated truncated mutants of IRF-4. The results showed that IRF-4 bound with LANA through its DNA-binding domain (residues 1–135) at the N-terminus (Figure 6C). The results which showed that in-vitro translated IRF-4 treated with DNase I still bound to LANA (Figure 6B), excluded the possibility that IRF-4 and LANA interaction was dependent on the DNA sequence. To confirm that these two molecules do associate in cells, we performed co-immunoprecipitation assays to determine if LANA forms a complex with IRF-4 in KSHV negative (DG75) and positive (BC3) B-lymphoma cell lines. The results of the co-IP assays from DG75 cells with exogenous LANA and IRF-4 co-expression, showed a complex of IRF-4 with LANA (Figure 7A, lane 6). Consistent with these results, the co-IP studies from BC3 cells showed that endogenous IRF-4 was dramatically immunoprecipitated with anti-LANA antibody, but not using a control mouse serum (Figure 7B, top and left panels). The reverse immunoprecipitation using anti-IRF-4 antibody further confirmed that LANA does form a complex with IRF-4 in KSHV infected cells (Figure 7B, top and right panels). This supports the data above that these two proteins can associate in the same molecular complex.

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