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The MHC-II transactivator CIITA inhibits Tat function and HIV-1 replication in human myeloid cells.

Forlani G, Turrini F, Ghezzi S, Tedeschi A, Poli G, Accolla RS, Tosi G - J Transl Med (2016)

Bottom Line: Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells.This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.

ABSTRACT

Background: We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells.

Methods: U937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants.

Results: CIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.

Conclusions: U937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1 replication. The generation of Plus-CIITA cells was instrumental to demonstrate the specific contribution of CIITA in terms of inhibition of Tat activity and HIV-1 restriction, independently from other cellular factors, including TRIM22. Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

No MeSH data available.


Related in: MedlinePlus

U937 Minus cells, but not U937 Plus cells express HLA-II and CIITA. a HLA-I and HLA-II phenotyping of U937 Minus and U937 Plus cells was carried out by immunofluorescence and FACS analysis. Raji B cells were used as a positive control for both HLA-I and HLA-II expression. Histograms represent fluorescence profiles of the cells indicated on the left, incubated with specific anti HLA-I or HLA-II mAbs (solid line) or with FITC-conjugated F(ab)2 anti mouse antibody (dashed line). By using three mAbs each covering subgroups (DQ1, DQ2 and DQ3) of DQ molecules we found that U937 Minus cells are DQ2-positive. Mean fluorescence (mf) values are expressed in the abscissa as arbitrary units (au). b HLA-II DR and CIITA mRNAs expression in U937 Minus cells, U937 Plus cells and Raji B cells was assessed by qRT-PCR. The results of a representative experiment performed in triplicates are shown. CIITA and HLA-II DR mRNA levels are expressed as values relative to those of Minus cells set to 1. Raji B cells were used as a positive control for both CIITA and HLA-II DR mRNA expression. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P = 0.0001). Error bars represent the standard deviation
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Fig1: U937 Minus cells, but not U937 Plus cells express HLA-II and CIITA. a HLA-I and HLA-II phenotyping of U937 Minus and U937 Plus cells was carried out by immunofluorescence and FACS analysis. Raji B cells were used as a positive control for both HLA-I and HLA-II expression. Histograms represent fluorescence profiles of the cells indicated on the left, incubated with specific anti HLA-I or HLA-II mAbs (solid line) or with FITC-conjugated F(ab)2 anti mouse antibody (dashed line). By using three mAbs each covering subgroups (DQ1, DQ2 and DQ3) of DQ molecules we found that U937 Minus cells are DQ2-positive. Mean fluorescence (mf) values are expressed in the abscissa as arbitrary units (au). b HLA-II DR and CIITA mRNAs expression in U937 Minus cells, U937 Plus cells and Raji B cells was assessed by qRT-PCR. The results of a representative experiment performed in triplicates are shown. CIITA and HLA-II DR mRNA levels are expressed as values relative to those of Minus cells set to 1. Raji B cells were used as a positive control for both CIITA and HLA-II DR mRNA expression. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P = 0.0001). Error bars represent the standard deviation

Mentions: To verify that the two U937 Plus and Minus isogenic cell clones differ for the HLA-II cell surface expression, we firstly assessed the complete HLA-II phenotype by immunofluorescence staining and FACS analysis. HLA-II DR was not expressed by U937 Plus cells, whereas it was expressed by U937 Minus cells although at lower levels compared to Raji B cell line (Fig. 1a). Similarly, HLA-II DP and HLA-II DQ2 were expressed in U937 Minus cells but not in U937 Plus cells. Conversely, both U937 cell clones expressed HLA class-I molecules on their cell surface (Fig. 1a). To verify whether the lack of HLA-II molecules in U937 Plus cells was due to a transcriptional defect, we measured the amount of HLA-II DR mRNA by qRT-PCR. According to the expression of HLA-II DR molecules, we detected HLA-II DR mRNA in Minus but not in Plus U937 cells (Fig. 1b). Thus, we concluded that the complete set of HLA-II molecules was not expressed on the surface of U937 Plus cells consequently to a block in HLA-II genes transcription. As HLA-II expression is regulated at transcriptional level by several factors, but is strictly dependent on the presence of CIITA, we next investigated whether the different HLA-II phenotype of the two U937 clones correlated with a different expression of CIITA. To this aim, we quantified CIITA mRNA levels in both U937 clones by qRT-PCR and found that only U937 Minus cells expressed CIITA mRNA, whereas U937 Plus cells did not (Fig. 1b). Overall, these data demonstrate that CIITA expression or lack of it marks a clear-cut distinction between U937 Minus (CIITA-positive) and U937 Plus (CIITA-negative) cells. In addition, these results indicated that this cellular model represented by isogenic cell clones naturally differing for CIITA expression is suitable to define the anti-viral role of this molecule in HIV-1 infection of myeloid cells.Fig. 1


The MHC-II transactivator CIITA inhibits Tat function and HIV-1 replication in human myeloid cells.

Forlani G, Turrini F, Ghezzi S, Tedeschi A, Poli G, Accolla RS, Tosi G - J Transl Med (2016)

U937 Minus cells, but not U937 Plus cells express HLA-II and CIITA. a HLA-I and HLA-II phenotyping of U937 Minus and U937 Plus cells was carried out by immunofluorescence and FACS analysis. Raji B cells were used as a positive control for both HLA-I and HLA-II expression. Histograms represent fluorescence profiles of the cells indicated on the left, incubated with specific anti HLA-I or HLA-II mAbs (solid line) or with FITC-conjugated F(ab)2 anti mouse antibody (dashed line). By using three mAbs each covering subgroups (DQ1, DQ2 and DQ3) of DQ molecules we found that U937 Minus cells are DQ2-positive. Mean fluorescence (mf) values are expressed in the abscissa as arbitrary units (au). b HLA-II DR and CIITA mRNAs expression in U937 Minus cells, U937 Plus cells and Raji B cells was assessed by qRT-PCR. The results of a representative experiment performed in triplicates are shown. CIITA and HLA-II DR mRNA levels are expressed as values relative to those of Minus cells set to 1. Raji B cells were used as a positive control for both CIITA and HLA-II DR mRNA expression. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P = 0.0001). Error bars represent the standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835826&req=5

Fig1: U937 Minus cells, but not U937 Plus cells express HLA-II and CIITA. a HLA-I and HLA-II phenotyping of U937 Minus and U937 Plus cells was carried out by immunofluorescence and FACS analysis. Raji B cells were used as a positive control for both HLA-I and HLA-II expression. Histograms represent fluorescence profiles of the cells indicated on the left, incubated with specific anti HLA-I or HLA-II mAbs (solid line) or with FITC-conjugated F(ab)2 anti mouse antibody (dashed line). By using three mAbs each covering subgroups (DQ1, DQ2 and DQ3) of DQ molecules we found that U937 Minus cells are DQ2-positive. Mean fluorescence (mf) values are expressed in the abscissa as arbitrary units (au). b HLA-II DR and CIITA mRNAs expression in U937 Minus cells, U937 Plus cells and Raji B cells was assessed by qRT-PCR. The results of a representative experiment performed in triplicates are shown. CIITA and HLA-II DR mRNA levels are expressed as values relative to those of Minus cells set to 1. Raji B cells were used as a positive control for both CIITA and HLA-II DR mRNA expression. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P = 0.0001). Error bars represent the standard deviation
Mentions: To verify that the two U937 Plus and Minus isogenic cell clones differ for the HLA-II cell surface expression, we firstly assessed the complete HLA-II phenotype by immunofluorescence staining and FACS analysis. HLA-II DR was not expressed by U937 Plus cells, whereas it was expressed by U937 Minus cells although at lower levels compared to Raji B cell line (Fig. 1a). Similarly, HLA-II DP and HLA-II DQ2 were expressed in U937 Minus cells but not in U937 Plus cells. Conversely, both U937 cell clones expressed HLA class-I molecules on their cell surface (Fig. 1a). To verify whether the lack of HLA-II molecules in U937 Plus cells was due to a transcriptional defect, we measured the amount of HLA-II DR mRNA by qRT-PCR. According to the expression of HLA-II DR molecules, we detected HLA-II DR mRNA in Minus but not in Plus U937 cells (Fig. 1b). Thus, we concluded that the complete set of HLA-II molecules was not expressed on the surface of U937 Plus cells consequently to a block in HLA-II genes transcription. As HLA-II expression is regulated at transcriptional level by several factors, but is strictly dependent on the presence of CIITA, we next investigated whether the different HLA-II phenotype of the two U937 clones correlated with a different expression of CIITA. To this aim, we quantified CIITA mRNA levels in both U937 clones by qRT-PCR and found that only U937 Minus cells expressed CIITA mRNA, whereas U937 Plus cells did not (Fig. 1b). Overall, these data demonstrate that CIITA expression or lack of it marks a clear-cut distinction between U937 Minus (CIITA-positive) and U937 Plus (CIITA-negative) cells. In addition, these results indicated that this cellular model represented by isogenic cell clones naturally differing for CIITA expression is suitable to define the anti-viral role of this molecule in HIV-1 infection of myeloid cells.Fig. 1

Bottom Line: Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells.This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.

ABSTRACT

Background: We previously demonstrated that the HLA class II transactivator CIITA inhibits HIV-1 replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the P-TEFb complex. Here, we analyzed the anti-viral function of CIITA in myeloid cells, another relevant HIV-1 target cell type. We sinvestigated clones of the U937 promonocytic cell line, either permissive (Plus) or non-permissive (Minus) to HIV-1 replication. This different phenotype has been associated with the expression of TRIM22 in U937 Minus but not in Plus cells.

Methods: U937 Plus cells stably expressing CIITA were generated and HLA-II positive clones were selected by cell sorting and cloning. HLA and CIITA proteins were analyzed by cytofluorometry and western blotting, respectively. HLA-II DR and CIITA mRNAs were quantified by qRT-PCR. Tat-dependent transactivation was assessed by performing the HIV-1 LTR luciferase gene reporter assay. Cells were infected with HIV-1 and viral replication was evaluated by measuring the RT activity in culture supernatants.

Results: CIITA was expressed only in HLA-II-positive U937 Minus cells, and this was strictly correlated with inhibition of Tat-dependent HIV-1 LTR transactivation in Minus but not in Plus cells. Overexpression of CIITA in Plus cells restored the suppression of Tat transactivation, confirming the inhibitory role of CIITA. Importantly, HIV-1 replication was significantly reduced in Plus-CIITA cells with respect to Plus parental cells. This effect was independent of TRIM22 as CIITA did not induce TRIM22 expression in Plus-CIITA cells.

Conclusions: U937 Plus and Minus cells represent an interesting model to study the role of CIITA in HIV-1 restriction in the monocytic/macrophage cell lineage. The differential expression of CIITA in CIITA-negative Plus and CIITA-positive Minus cells correlated with their capacity to support or not HIV-1 replication, respectively. In Minus cells CIITA targeted the viral transactivator Tat to inhibit HIV-1 replication. The generation of Plus-CIITA cells was instrumental to demonstrate the specific contribution of CIITA in terms of inhibition of Tat activity and HIV-1 restriction, independently from other cellular factors, including TRIM22. Thus, CIITA acts as a general restriction factor against HIV-1 not only in T cells but also in myeloid cells.

No MeSH data available.


Related in: MedlinePlus