Abstract
BackgroundWe 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.MethodsU937 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.ResultsCIITA 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.ConclusionsU937 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.
Highlights
We previously demonstrated that the human leucocytes antigens (HLA) class II transactivator CIITA inhibits human immunodeficiency virus-1 (HIV-1) replication in T cells by competing with the viral transactivator Tat for the binding to Cyclin T1 subunit of the Positive Transcription Elongation Factor b (P-TEFb) complex
Lack of CIITA expression is responsible for the HLA‐II‐negative phenotype of U937 Plus cells 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 fluorescence activated cell sorter (FACS) analysis
HLAII DR was not expressed by U937 Plus cells, whereas it was expressed by U937 Minus cells at lower levels compared to Raji B cell line (Fig. 1a)
Summary
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. These reservoirs are resistant even to combination anti-retroviral therapy (cART) and are responsible for the ignition of viral replication and disease progression at therapy suspension [4] Myeloid cells such as dendritic cells (DC) and tissue macrophages play a crucial role in HIV-1 primary infection by capturing the virus through mucosal transmission [5] and subsequently by promoting virus dissemination to Th lymphocytes [6]. Circulating monocytes, being CD4 and CCR5 positive, are less permissive to the virus than their differentiated macrophage counterpart, likely because of high levels of basal expression of restriction factors such as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like A (APOBEC3A) [10, 11] In this regard, many restriction factors have been discovered in last decade which inhibit different steps of the viral life cycle from capsid uncoating to viral budding. The importance of restriction factors is highlighted by the observation that HIV-1 has evolved the so-called “accessory proteins”, such as Nef, Vif, Vpu and, in case of HIV-2, VpX, to antagonize the antiviral effects of host restriction factors [13]
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