Forlani Greta, Turrini Filippo, Ghezzi Silvia, Tedeschi Alessandra, Poli Guido, Accolla Roberto S, Tosi Giovanna
Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy.
Viral Pathogens and Biosafety Unit San Raffaele Scientific Institute, Milan, Italy.
J Transl Med. 2016 Apr 18;14:94. doi: 10.1186/s12967-016-0853-5.
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.
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.
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.
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.
我们之前证明，HLA II类反式激活因子CIITA通过与病毒反式激活因子Tat竞争结合P-TEFb复合物的细胞周期蛋白T1亚基，抑制T细胞中的HIV-1复制。在此，我们分析了CIITA在髓系细胞（另一种相关的HIV-1靶细胞类型）中的抗病毒功能。我们研究了U937原单核细胞系的克隆，这些克隆对HIV-1复制要么是允许的（阳性），要么是不允许的（阴性）。这种不同的表型与U937阴性细胞而非阳性细胞中TRIM22的表达有关。
生成稳定表达CIITA的U937阳性细胞，并通过细胞分选和克隆选择HLA-II阳性克隆。分别通过细胞荧光测定法和蛋白质印迹法分析HLA和CIITA蛋白。通过qRT-PCR定量HLA-II DR和CIITA mRNA。通过进行HIV-1 LTR荧光素酶基因报告基因测定评估Tat依赖性反式激活。用HIV-1感染细胞，并通过测量培养上清液中的RT活性评估病毒复制。
CIITA仅在HLA-II阳性的U937阴性细胞中表达，这与阴性而非阳性细胞中Tat依赖性HIV-1 LTR反式激活的抑制严格相关。CIITA在阳性细胞中的过表达恢复了对Tat反式激活的抑制，证实了CIITA的抑制作用。重要的是，与阳性亲本细胞相比，阳性-CIITA细胞中的HIV-1复制显著减少。由于CIITA在阳性-CIITA细胞中不诱导TRIM22表达，所以这种作用与TRIM22无关。
U937阳性和阴性细胞是研究CIITA在单核细胞/巨噬细胞谱系中HIV-1限制作用的有趣模型。CIITA在CIITA阴性的阳性细胞和CIITA阳性的阴性细胞中的差异表达分别与其支持或不支持HIV-1复制的能力相关。在阴性细胞中，CIITA靶向病毒反式激活因子Tat以抑制HIV-1复制。阳性-CIITA细胞的产生有助于证明CIITA在抑制Tat活性和HIV-1限制方面的特定贡献，独立于包括TRIM22在内的其他细胞因子。因此，CIITA不仅在T细胞中，而且在髓系细胞中都作为一种针对HIV-1的一般限制因子发挥作用。
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