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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 stably expressed in U937 Plus cells inhibits HIV-1 replication without inducing TRIM22 transcription. a U937 Minus cells, U937 Plus cells and U937 Plus-CIITA 1F6 clone were infected with HIV-1IIIB/LAI and viral replication was measured by monitoring RT activity in the culture supernatants up to 41 days post-infection (pi) Mean values of triplicate cultures are shown. The data are representative of four independent experiments. b At 24 h pi the amount of HIV-1 proviral DNA was assessed by real time PCR by using primers specific for gag. c TRIM22 mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. U937 Minus cells TRIM22 mRNA level was set to 1. d U937 Minus cells either untreated (−) or treated (+) with 10 nM vitamin D3 for 72 h were analyzed for CIITA and HLA-II DR mRNAs expression by qRT-PCR. In Vitamin D3-untreated U937 Minus cells the amount of both CIITA and HLA-II DR mRNAs was set to 1. Results are the average of three independent experiments (***P = 0.0001; **P = 0.0094, unpaired two-tailed t test). Error bars show standard deviation
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Fig5: CIITA stably expressed in U937 Plus cells inhibits HIV-1 replication without inducing TRIM22 transcription. a U937 Minus cells, U937 Plus cells and U937 Plus-CIITA 1F6 clone were infected with HIV-1IIIB/LAI and viral replication was measured by monitoring RT activity in the culture supernatants up to 41 days post-infection (pi) Mean values of triplicate cultures are shown. The data are representative of four independent experiments. b At 24 h pi the amount of HIV-1 proviral DNA was assessed by real time PCR by using primers specific for gag. c TRIM22 mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. U937 Minus cells TRIM22 mRNA level was set to 1. d U937 Minus cells either untreated (−) or treated (+) with 10 nM vitamin D3 for 72 h were analyzed for CIITA and HLA-II DR mRNAs expression by qRT-PCR. In Vitamin D3-untreated U937 Minus cells the amount of both CIITA and HLA-II DR mRNAs was set to 1. Results are the average of three independent experiments (***P = 0.0001; **P = 0.0094, unpaired two-tailed t test). Error bars show standard deviation

Mentions: To assess whether the CIITA-mediated inhibition of Tat function observed in CIITA-positive U937 cells was correlated with an inhibition of HIV-1 replication we infected the different U937 clones with HIV-1IIIB/LAI and measured the levels of RT viral activity in the culture supernatants up to 41 days post-infection. As previously shown [32, 34], HIV-1 replicates efficiently in U937 Plus cells reaching a peak at day 27 (Fig. 5a, solid lane, open circle). In contrast, virus replication in U937 Minus cells was not detected in this time frame (Fig. 5a, solid lane, solid square), consistently with previous publications [34]. Of note, in CIITA-expressing U937 Plus clone 1F6 the HIV-1 replication was significantly inhibited with respect to U937 Plus cells (Fig. 5a, dashed lane, solid triangle). In particular, at day 27 the RT activity measured in U937 Plus-CIITA 1F6 clone was less than 40 % the one measured in U937 Plus cells (6.318 and 16.534 cpm/μl, respectively). This different profile of virus replication was not accounted for by a different infection efficiency in that the amount of HIV-1 DNA detected 24 h post-infection was comparable in all tested cells (Fig. 5b). In conclusion, the results obtained by infecting U937 Plus-CIITA clone 1F6, indicate that CIITA contributes, at least in part, in determining the non-permissive phenotype of U937 Minus cells by inhibiting Tat activity. In U937 Plus-CIITA 1F6 clone, HIV-1 replication was significantly suppressed compared with Plus parental cells, but did not reach the inhibition level observed in Minus cells (Fig. 5a). This result let us to hypothesize that other host factors with anti-viral functions may contribute to the non-permissive phenotype of Minus cells. Among these, TRIM22, expressed in U937 Minus cells, but not in U937 Plus cells, was shown to inhibit basal as well as PMA plus ionomycin-induced HIV-1 transcription independently of Tat and NF-kB [34]. Therefore, we verified whether CIITA transfection in Plus cells induced TRIM22 mRNA as quantified by qRT-PCR. We found that only U937 Minus cells expressed TRIM22 mRNA whereas CIITA-positive 1C11 and 1F6 clones as well as the CIITA-negative 4G2 clone did not (Fig. 5c). Thus, in CIITA-expressing U937 Plus clones, the impairment of HIV-1 replication is solely due to CIITA and not to the induction of TRIM22 expression.Fig. 5


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 stably expressed in U937 Plus cells inhibits HIV-1 replication without inducing TRIM22 transcription. a U937 Minus cells, U937 Plus cells and U937 Plus-CIITA 1F6 clone were infected with HIV-1IIIB/LAI and viral replication was measured by monitoring RT activity in the culture supernatants up to 41 days post-infection (pi) Mean values of triplicate cultures are shown. The data are representative of four independent experiments. b At 24 h pi the amount of HIV-1 proviral DNA was assessed by real time PCR by using primers specific for gag. c TRIM22 mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. U937 Minus cells TRIM22 mRNA level was set to 1. d U937 Minus cells either untreated (−) or treated (+) with 10 nM vitamin D3 for 72 h were analyzed for CIITA and HLA-II DR mRNAs expression by qRT-PCR. In Vitamin D3-untreated U937 Minus cells the amount of both CIITA and HLA-II DR mRNAs was set to 1. Results are the average of three independent experiments (***P = 0.0001; **P = 0.0094, unpaired two-tailed t test). Error bars show standard deviation
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Fig5: CIITA stably expressed in U937 Plus cells inhibits HIV-1 replication without inducing TRIM22 transcription. a U937 Minus cells, U937 Plus cells and U937 Plus-CIITA 1F6 clone were infected with HIV-1IIIB/LAI and viral replication was measured by monitoring RT activity in the culture supernatants up to 41 days post-infection (pi) Mean values of triplicate cultures are shown. The data are representative of four independent experiments. b At 24 h pi the amount of HIV-1 proviral DNA was assessed by real time PCR by using primers specific for gag. c TRIM22 mRNA expression was assessed by qRT-PCR in U937 Minus cells, U937 Plus cells and in clones 1C11, 1F6, 4G2. U937 Minus cells TRIM22 mRNA level was set to 1. d U937 Minus cells either untreated (−) or treated (+) with 10 nM vitamin D3 for 72 h were analyzed for CIITA and HLA-II DR mRNAs expression by qRT-PCR. In Vitamin D3-untreated U937 Minus cells the amount of both CIITA and HLA-II DR mRNAs was set to 1. Results are the average of three independent experiments (***P = 0.0001; **P = 0.0094, unpaired two-tailed t test). Error bars show standard deviation
Mentions: To assess whether the CIITA-mediated inhibition of Tat function observed in CIITA-positive U937 cells was correlated with an inhibition of HIV-1 replication we infected the different U937 clones with HIV-1IIIB/LAI and measured the levels of RT viral activity in the culture supernatants up to 41 days post-infection. As previously shown [32, 34], HIV-1 replicates efficiently in U937 Plus cells reaching a peak at day 27 (Fig. 5a, solid lane, open circle). In contrast, virus replication in U937 Minus cells was not detected in this time frame (Fig. 5a, solid lane, solid square), consistently with previous publications [34]. Of note, in CIITA-expressing U937 Plus clone 1F6 the HIV-1 replication was significantly inhibited with respect to U937 Plus cells (Fig. 5a, dashed lane, solid triangle). In particular, at day 27 the RT activity measured in U937 Plus-CIITA 1F6 clone was less than 40 % the one measured in U937 Plus cells (6.318 and 16.534 cpm/μl, respectively). This different profile of virus replication was not accounted for by a different infection efficiency in that the amount of HIV-1 DNA detected 24 h post-infection was comparable in all tested cells (Fig. 5b). In conclusion, the results obtained by infecting U937 Plus-CIITA clone 1F6, indicate that CIITA contributes, at least in part, in determining the non-permissive phenotype of U937 Minus cells by inhibiting Tat activity. In U937 Plus-CIITA 1F6 clone, HIV-1 replication was significantly suppressed compared with Plus parental cells, but did not reach the inhibition level observed in Minus cells (Fig. 5a). This result let us to hypothesize that other host factors with anti-viral functions may contribute to the non-permissive phenotype of Minus cells. Among these, TRIM22, expressed in U937 Minus cells, but not in U937 Plus cells, was shown to inhibit basal as well as PMA plus ionomycin-induced HIV-1 transcription independently of Tat and NF-kB [34]. Therefore, we verified whether CIITA transfection in Plus cells induced TRIM22 mRNA as quantified by qRT-PCR. We found that only U937 Minus cells expressed TRIM22 mRNA whereas CIITA-positive 1C11 and 1F6 clones as well as the CIITA-negative 4G2 clone did not (Fig. 5c). Thus, in CIITA-expressing U937 Plus clones, the impairment of HIV-1 replication is solely due to CIITA and not to the induction of TRIM22 expression.Fig. 5

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