MCPIP1 restricts HIV infection and is rapidly degraded in activated CD4+ T cells

Abstract

HIV-1 primarily infects activated CD4+ T cells and macrophages. Quiescent CD4+ T cells, however, possess cellular factors that limit HIV-1 infection at different postentry steps of the viral life cycle. Here, we show that the previously reported immune regulator monocyte chemotactic protein-induced protein 1 (MCPIP1) restricts HIV-1 production in CD4+ T cells. While the ectopic expression of MCPIP1 in cell lines abolished the production of HIV-1, silencing of MCPIP1 enhanced HIV-1 production. Subsequent analysis indicated that MCPIP1 imposes its restriction by decreasing the steady levels of viral mRNA species through its RNase domain. Remarkably, common T-cell stimuli induced the rapid degradation of MCPIP1 in both T-cell lines and quiescent human CD4+ T cells. Lastly, blocking the proteosomal degradation of MCPIP1 by MG132 abrogated HIV-1 production in phorbol 12-myristate 13-acetate/ionomycin-stimulated human CD4+ T cells isolated from healthy donors. Overall, MCPIP1 poses a potent barrier against HIV-1 infection at a posttranscriptional stage. Although the observed HIV restriction conferred by MCPIP1 does not seem to be overcome by any viral protein, it is removed during cellular stimulation. These findings provide insights into the mechanisms of cellular activation-mediated HIV-1 production in CD4+ T cells.

Keywords: antiviral immunity; restriction factor.

Fig. 1.

MCPIP1/ZC3H12A potently inhibits HIV-1 in both transfection and infection systems. (A and B) HIV-1 pNL4.3 proviral construct (0.2 μg) was cotransfected into 293T cells with 0, 0.1, 0.2, and 0.5 μg indicated plasmids. At 48 h posttransfection, supernatants were collected for ELISA to determine the secretion of HIV-1 p24 (capsid protein) or TZM-bl assay to measure the production of infectious virus (B). (C) Stable cells derived from CEM-SS (a human T-cell line that is highly permissive to HIV infection) expressing GFP or GFP-MCPIP1 were inoculated with HIV-1 (NL4.3 strain) at two doses (input measured as 10 or 20 ng p24) for 2 h. After extensive wash, cells were further incubated, and supernatants were collected at days 2, 3, 4, and 5 postinfection before TZM-bl assay. (Inset) Western blot image indicates the expression of MCPIP1 in stable cells. (D) Similar assays were performed as in A, except that p89.6 (dual tropic), pNL(AD8) (M tropic), and pLAI.2 (T tropic) proviral DNA were included. A, C, and D are representatives of three independent experiments; B is an average of five independent experiments. A thymidine kinase promoter-driven Renilla luciferase reporter construct was included in all transfection experiments to control for transfection efficiency. (E) MT2 and MT4 human T-cell lines were induced to express GFP-MCPIP1 and then infected by NL4.3 virus. At 48 h postinfection, supernatants were collected for determination of the production of infectious virus and the release of p24. (F) CEM-SS cells were transfected with (Upper) three siRNAs (A, B, and C) by electroporation to knock down the endogenous MCPIP1 (Western blot). The production of infectious HIV-1 from the cells was measured by TZM-bl assays. RLU, relative light unit.

Fig. 2.

MCPIP1 inhibits HIV-1 at a posttranscription step. CEM-SS or HEK293T cells were generated to express GFP-tagged MCPIP1 or GFP alone under the control of doxycycline (Dox). (A) CEM-SS cells were incubated with 5 ng/mL Dox overnight to induce GFP-MCPIP1 or GFP and then infected with various amounts of HIV NL4.3; 48 h later, the culture supernatants were analyzed for the infectivity measured by TZM assay. In B, the comparative number of viral RNA was quantified by real-time RT-PCR. An equivalent amount of HCV was added during RNA extraction for normalization. (C) The viral proteins in whole-cell lysates were analyzed by immunoblotting against gp120, gag, nef, and the Dox-induced GFP-tagged MCPIP1. (D) 2-LTR circles were comparatively quantified by real-time PCR. The relative quantification of provirus was performed by Alu-PCR. The level of mtDNA served as a normalization control. (E) The pGL3 reporter containing LTR or UTR (LTR + PBS + Psi) was transiently cotransfected into HEK293T cells with construct expressing MCPIP1 or control vector. The TK promoter-driven vector constitutive expressing Renilla luciferase was used as internal control. The Firefly and Renilla luciferase activities were measured 24 h after transfection. The ratio of Firefly to Renilla is displayed. (F) The CEM-SS cells were inoculated with HIV-1 for 24 h. After being washed, the cells were induced with various amount of Dox to express GFP-tagged MCPIP1 or GFP. At 48 h postinduction, the infectivity of culture supernatant was quantified by TZM assay. The corresponding levels of GFP-tagged MCPIP1 were measured by immunoblotting. (G) The same analysis was done with pNL4.3-transfected 293 cells. HEK293 cells were transiently transfected with pNL4.3. At 24 h posttransfection, cells were induced with various amount of Dox to express GFP-tagged MCPIP1 or GFP. At 48 h postinduction, the infectivity of culture supernatant was quantified by TZM assay. The corresponding level of GFP-tagged MCPIP1 was measured by immunoblotting. RLU, relative light unit.

Fig. 3.

The RNase activity of MCPIP1 is required for its anti-HIV activity. (A) The viral RNA was extracted from HIV-infected CEM-Dox^ON-GFP-MCPIP1 cells [in the presence or absence of doxycycline (Dox)] and subjected to Northern blot analysis for detection of viral RNA species. (Top) Unspliced ∼9-kb mRNAs. (Middle) Singly or incompletely spliced ∼4-kb mRNAs. (Bottom) Multiply spliced ∼1.8-kb mRNAs. The 28S and 18S rRNAs were included as loading controls. (B) CEM-SS cells were electroporated with siRNA against MCPIP1 (construct B) or scrambled siRNA (si-CTRL) at 100 nM. Two days postelectroporation, cells were inoculated with HIV NL4.3. After an additional 2 d of incubation, cellular RNA were isolated and subjected to quantitative RT-PCR analysis. Upper shows the relative fold of change over si-CTRL (HIV Gag, Vif, and Tat-rev mRNA), and Lower shows the knockdown of endogenous MCPIP1. (C, Top) A schematic diagram for point mutations in the putative functional conserved domains of MCPIP1. D141N mutation abolished both deubiquinating (DUB) and RNase activity; D225/226A lost RNase activity but maintained DUB activity, whereas C306R mutation at the CCCH region lost the DUB activity (21). (Middle) HEK293T cells were transiently cotransfected with pNL4.3 and constructs expressing the indicated mutant form of Flag-tagged MCPIP1; 48 h later, the infectivity of the culture supernatant was measured by TZM assay. (Bottom) CEM-SS cells were also generated to express these mutant forms of MCPIP1 under the control of Dox. After 24 h of incubation with Dox, the cells were infected with HIV; 48 h later, the infectivity of the culture supernatant was measured by TZM assay. (D) The same experiments were done with domain-deleted forms of MCPIP1 (Middle, HEK293 cells; Bottom, CEM-SS cells). P < 0.005 (n = 3, SD). RLU, relative light unit.

Fig. 4.

MCPIP1 is rapidly degraded on T-cell stimulation. (A) CEM-SS cells were infected with HIV for several days. Endogenous MCPIP1 was detected by Western blotting. (B) CEM-SS T cells were treated with PMA/Iono or anti-CD3 and -CD28 antibodies for 1 or 2 d. Endogenous MCPIP1 was detected by Western blotting. (C) Primary human PBMCs and purified CD4+ T cells were stimulated with PMA/Iono for 6 or 24 h. (D) CEM-SS cells expressing GFP-MCPIP1 were cultured in the presence or absence of MG132 for 1 h and then stimulated with PMA/Iono for 18 h. (E) CD4+ T cells from three donors were nucleofected with pNL4.3 and then treated with MG132 for 1 h followed by stimulation of PMA/Iono for 24 h before collection of supernatants. The amount of released p24 was measured by ELISA.