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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

CIITA inhibits Tat transactivating function in U937 Minus cells and in U937 Plus-CIITA clones. U937 Minus (black columns) and U937 Plus (gray columns) cells were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV, and increasing amounts (10, 20 ng) of plasmid coding for Tat (pcTat). CIITA-positive Plus clones 1C11, 1F6 and CIITA-negative Plus 4G2 clone were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV and 20 ng of pcTat. The basal HIV-1 LTR activity was set to 1 for each cell (−). Mean luciferase values, normalized to Renilla values, are presented as fold of activation (Tat-dependent vs basal). Results are the average of two independent experiments (**P < 0.005, unpaired two-tailed t test). Error bars show standard deviation
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Fig3: CIITA inhibits Tat transactivating function in U937 Minus cells and in U937 Plus-CIITA clones. U937 Minus (black columns) and U937 Plus (gray columns) cells were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV, and increasing amounts (10, 20 ng) of plasmid coding for Tat (pcTat). CIITA-positive Plus clones 1C11, 1F6 and CIITA-negative Plus 4G2 clone were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV and 20 ng of pcTat. The basal HIV-1 LTR activity was set to 1 for each cell (−). Mean luciferase values, normalized to Renilla values, are presented as fold of activation (Tat-dependent vs basal). Results are the average of two independent experiments (**P < 0.005, unpaired two-tailed t test). Error bars show standard deviation

Mentions: We have previously demonstrated that CIITA inhibits HIV-1 replication in human T cells by interfering with Tat function [25]. Therefore, we investigated whether the HIV-1 permissive and non-permissive phenotypes of U937 Plus and Minus cells, respectively, were due to their capacity to support or inhibit Tat-dependent HIV-1 LTR transactivation as a consequence of their different expression of CIITA. To this aim, we measured the Tat-dependent luciferase activities in U937 Minus, Plus and Plus-CIITA cells transiently transfected with the pHIV-1LTR-Luc reporter gene and increasing amounts of Tat expressing vector. An efficient Tat-dependent transactivation of HIV-1 transcription was observed in U937 Plus cells, whereas it was significantly reduced in the U937 Minus cells at both doses of Tat (Fig. 3, grey and black columns, respectively). Remarkably, in U937 Plus-CIITA clones 1C11 and 1F6 transfected with the highest amount of Tat plasmid, Tat transactivation is inhibited similarly to U937 Minus cells. In the 4G2 cell clone devoid of CIITA expression, Tat-mediated HIV-1 LTR transactivation was unaffected (Fig. 3). These results indicate that the permissive and the non-permissive phenotypes to HIV-1 infection of U937 Plus and Minus cells correlate with their capacity or incapacity, respectively, to support Tat-mediated HIV-1 LTR transcription. Importantly, the stable expression of CIITA in Plus cells abrogated Tat transactivating function. Thus, CIITA plays a major role in the inhibition of Tat transactivation, not only in T cells but also in myeloid cells, at least in the cellular model here investigated.Fig. 3


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)

CIITA inhibits Tat transactivating function in U937 Minus cells and in U937 Plus-CIITA clones. U937 Minus (black columns) and U937 Plus (gray columns) cells were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV, and increasing amounts (10, 20 ng) of plasmid coding for Tat (pcTat). CIITA-positive Plus clones 1C11, 1F6 and CIITA-negative Plus 4G2 clone were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV and 20 ng of pcTat. The basal HIV-1 LTR activity was set to 1 for each cell (−). Mean luciferase values, normalized to Renilla values, are presented as fold of activation (Tat-dependent vs basal). Results are the average of two independent experiments (**P < 0.005, unpaired two-tailed t test). Error bars show standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835826&req=5

Fig3: CIITA inhibits Tat transactivating function in U937 Minus cells and in U937 Plus-CIITA clones. U937 Minus (black columns) and U937 Plus (gray columns) cells were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV, and increasing amounts (10, 20 ng) of plasmid coding for Tat (pcTat). CIITA-positive Plus clones 1C11, 1F6 and CIITA-negative Plus 4G2 clone were co-transfected with fixed amount of pHIV-1 LTR-Luc, phRL-CMV and 20 ng of pcTat. The basal HIV-1 LTR activity was set to 1 for each cell (−). Mean luciferase values, normalized to Renilla values, are presented as fold of activation (Tat-dependent vs basal). Results are the average of two independent experiments (**P < 0.005, unpaired two-tailed t test). Error bars show standard deviation
Mentions: We have previously demonstrated that CIITA inhibits HIV-1 replication in human T cells by interfering with Tat function [25]. Therefore, we investigated whether the HIV-1 permissive and non-permissive phenotypes of U937 Plus and Minus cells, respectively, were due to their capacity to support or inhibit Tat-dependent HIV-1 LTR transactivation as a consequence of their different expression of CIITA. To this aim, we measured the Tat-dependent luciferase activities in U937 Minus, Plus and Plus-CIITA cells transiently transfected with the pHIV-1LTR-Luc reporter gene and increasing amounts of Tat expressing vector. An efficient Tat-dependent transactivation of HIV-1 transcription was observed in U937 Plus cells, whereas it was significantly reduced in the U937 Minus cells at both doses of Tat (Fig. 3, grey and black columns, respectively). Remarkably, in U937 Plus-CIITA clones 1C11 and 1F6 transfected with the highest amount of Tat plasmid, Tat transactivation is inhibited similarly to U937 Minus cells. In the 4G2 cell clone devoid of CIITA expression, Tat-mediated HIV-1 LTR transactivation was unaffected (Fig. 3). These results indicate that the permissive and the non-permissive phenotypes to HIV-1 infection of U937 Plus and Minus cells correlate with their capacity or incapacity, respectively, to support Tat-mediated HIV-1 LTR transcription. Importantly, the stable expression of CIITA in Plus cells abrogated Tat transactivating function. Thus, CIITA plays a major role in the inhibition of Tat transactivation, not only in T cells but also in myeloid cells, at least in the cellular model here investigated.Fig. 3

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