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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

Cellular and molecular characterization of U937 Plus cells stably expressing exogenous CIITA. a U937 Plus cells were stably transfected with pcfCIITA vector expressing flag-tagged CIITA, generating Plus-CIITA cells. Bulk population was analyzed for HLA-II DR expression by FACS analysis. HLA-II-positive clones were isolated by sorting and cloning. FITC-conjugated isotype-matched secondary antibody (IgG2a) (dotted line) represents the negative control. b HLA-II DR mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. The results of a representative experiment performed in triplicates are shown. mRNA values are expressed relatively to that of Minus cells set to 1. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P < 0.005). Error bars represent the standard deviation. c Cell lysates obtained from U937 Plus cells (60 × 106 cells) (lane 1), U937 Minus cells (60 × 106 cells) (lane 2) and CIITA-transfected Plus clones (30 × 106 cells) (lanes 3–5) were immunoprecipitated with an anti-CIITA antibody (IP a CIITA) and analyzed for the presence of CIITA by western blotting. Raji cell lysate (30 × 106 cells) was used as a positive control of CIITA expression (lane 6). Eight percent of the pre-cleared cell lysates was analyzed for the expression of α-tubulin by western blotting (input)
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Fig2: Cellular and molecular characterization of U937 Plus cells stably expressing exogenous CIITA. a U937 Plus cells were stably transfected with pcfCIITA vector expressing flag-tagged CIITA, generating Plus-CIITA cells. Bulk population was analyzed for HLA-II DR expression by FACS analysis. HLA-II-positive clones were isolated by sorting and cloning. FITC-conjugated isotype-matched secondary antibody (IgG2a) (dotted line) represents the negative control. b HLA-II DR mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. The results of a representative experiment performed in triplicates are shown. mRNA values are expressed relatively to that of Minus cells set to 1. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P < 0.005). Error bars represent the standard deviation. c Cell lysates obtained from U937 Plus cells (60 × 106 cells) (lane 1), U937 Minus cells (60 × 106 cells) (lane 2) and CIITA-transfected Plus clones (30 × 106 cells) (lanes 3–5) were immunoprecipitated with an anti-CIITA antibody (IP a CIITA) and analyzed for the presence of CIITA by western blotting. Raji cell lysate (30 × 106 cells) was used as a positive control of CIITA expression (lane 6). Eight percent of the pre-cleared cell lysates was analyzed for the expression of α-tubulin by western blotting (input)

Mentions: Although U937 cell clones differ for the expression of CIITA, we cannot exclude that other factors could contribute to their divergent capacity to sustain productive HIV-1 infection. Thus, to clarify the role of CIITA in HIV-1 restriction we generated U937 Plus cells expressing exogenous CIITA. The expression vector of flag epitope-tagged CIITA was stably transfected in the CIITA-negative U937 Plus parental cells. Transfected cells were selected with antibiotics and analyzed by immunofluorescence and cytofluorometry for HLA-II DR expression. A large proportion of cells expressed HLA-DR, demonstrating that CIITA alone was able to induce the expression of HLA-II genes (Fig. 2a). Highly expressing HLA-II DR cells were isolated by limiting dilution cloning and two HLA-II DR-positive clones (1C11, 1F6) and one negative clone (4G2) were selected for subsequent analyses (Fig. 2a). According to their HLA-II DR cell surface expression, the U937 cell clones 1C11 and 1F6 expressed higher levels of HLA-II DR mRNA compared to U937 Minus cells.Fig. 2


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)

Cellular and molecular characterization of U937 Plus cells stably expressing exogenous CIITA. a U937 Plus cells were stably transfected with pcfCIITA vector expressing flag-tagged CIITA, generating Plus-CIITA cells. Bulk population was analyzed for HLA-II DR expression by FACS analysis. HLA-II-positive clones were isolated by sorting and cloning. FITC-conjugated isotype-matched secondary antibody (IgG2a) (dotted line) represents the negative control. b HLA-II DR mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. The results of a representative experiment performed in triplicates are shown. mRNA values are expressed relatively to that of Minus cells set to 1. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P < 0.005). Error bars represent the standard deviation. c Cell lysates obtained from U937 Plus cells (60 × 106 cells) (lane 1), U937 Minus cells (60 × 106 cells) (lane 2) and CIITA-transfected Plus clones (30 × 106 cells) (lanes 3–5) were immunoprecipitated with an anti-CIITA antibody (IP a CIITA) and analyzed for the presence of CIITA by western blotting. Raji cell lysate (30 × 106 cells) was used as a positive control of CIITA expression (lane 6). Eight percent of the pre-cleared cell lysates was analyzed for the expression of α-tubulin by western blotting (input)
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Related In: Results  -  Collection

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Fig2: Cellular and molecular characterization of U937 Plus cells stably expressing exogenous CIITA. a U937 Plus cells were stably transfected with pcfCIITA vector expressing flag-tagged CIITA, generating Plus-CIITA cells. Bulk population was analyzed for HLA-II DR expression by FACS analysis. HLA-II-positive clones were isolated by sorting and cloning. FITC-conjugated isotype-matched secondary antibody (IgG2a) (dotted line) represents the negative control. b HLA-II DR mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. The results of a representative experiment performed in triplicates are shown. mRNA values are expressed relatively to that of Minus cells set to 1. Unpaired two-tailed t test has been performed (****P < 0.0001; ***P < 0.005). Error bars represent the standard deviation. c Cell lysates obtained from U937 Plus cells (60 × 106 cells) (lane 1), U937 Minus cells (60 × 106 cells) (lane 2) and CIITA-transfected Plus clones (30 × 106 cells) (lanes 3–5) were immunoprecipitated with an anti-CIITA antibody (IP a CIITA) and analyzed for the presence of CIITA by western blotting. Raji cell lysate (30 × 106 cells) was used as a positive control of CIITA expression (lane 6). Eight percent of the pre-cleared cell lysates was analyzed for the expression of α-tubulin by western blotting (input)
Mentions: Although U937 cell clones differ for the expression of CIITA, we cannot exclude that other factors could contribute to their divergent capacity to sustain productive HIV-1 infection. Thus, to clarify the role of CIITA in HIV-1 restriction we generated U937 Plus cells expressing exogenous CIITA. The expression vector of flag epitope-tagged CIITA was stably transfected in the CIITA-negative U937 Plus parental cells. Transfected cells were selected with antibiotics and analyzed by immunofluorescence and cytofluorometry for HLA-II DR expression. A large proportion of cells expressed HLA-DR, demonstrating that CIITA alone was able to induce the expression of HLA-II genes (Fig. 2a). Highly expressing HLA-II DR cells were isolated by limiting dilution cloning and two HLA-II DR-positive clones (1C11, 1F6) and one negative clone (4G2) were selected for subsequent analyses (Fig. 2a). According to their HLA-II DR cell surface expression, the U937 cell clones 1C11 and 1F6 expressed higher levels of HLA-II DR mRNA compared to U937 Minus cells.Fig. 2

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