Human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells - PubMed (original) (raw)

Human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells

Brian P Doehle et al. J Virol. 2009 Oct.

Abstract

Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication, but these defenses fail to suppress human immunodeficiency virus (HIV) infection, which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here, we examined antiviral defenses in CD4+ cells during virus infection and coinfection, revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression, but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells, which supported robust viral replication, whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections.

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Figures

FIG. 1.

IRF-3 levels are reduced in response to HIV-1 infection. (A) SupT1 cells or PBMCs were infected with HIV-1LAI (MOI of 10). Cells were harvested at the indicated times postinfection and probed by immunoblotting to determine expression levels of ISG15, ISG56, p24, and IRF-3. Actin levels were monitored to control for protein leading. Where indicated, cells were infected with 50 hemagglutinating units/ml of SenV. (B) HeLa CD4+ cells were cultured in chamber slides and infected as described above. Cells were fixed and triple stained by using antibodies against IRF-3 and HIV p24 and by staining nuclei with DAPI. Cells were visualized by immunofluorescence microscopy. IRF-3 is shown in red, p24 is shown in green, and nuclei are shown in blue. (C) THP1 cells were infected with HIV-1JR-CSF at an MOI of 1, and infection was allowed to progress for the indicated amounts of time. Cells were harvested, and extracts were subjected to immunoblot analysis to measure the levels of IRF-3, HIV p24, and actin. Values represent the percentages of IRF-3 compared to that at time zero. Data shown are representative of three independent experiments. hpi, hours postinfection.

FIG. 2.

IRF-3 degradation by HIV-1 supports evasion of innate defenses that limit infection. (A) SupT1 cells were mock infected or infected with HIV-1LAI at an MOI of 1. Cells were harvested at the indicated times for immunoblot analysis of IRF-7, IRF-3, HIV p24, and actin levels. Values represent the percentages of IRF-3 compared to that at time zero of infection. (B) IRF-3 mRNA levels were determined by qPCR analysis. SupT1 cells were infected as described above. Total RNA was recovered, and qPCR was performed using specific primers for IRF-3 and GAPDH mRNA (for normalization) (n = 3). (C) SupT1 cells were mock infected or infected with HIV-1LAI. Cells were harvested 24 h later, or CHX was added to the cultures and the cells were harvested 28, 32, or 48 h after HIV-1 infection (corresponding to 4, 8, or 24 h of CHX treatment, respectively). IRF-3, IRF-7, and actin levels were measured by immunoblot assay. Densitometry was used to quantify the amount of each protein species at each time postinfection and posttreatment, from which the half-life for each molecule was determined. The graph shows the relative percentage of IRF-3 remaining at each time point for mock- or HIV-1-infected cultures compared to that at time zero for mock-infected cells. (D) SupT1 cells were transfected with a plasmid expressing IRF-3(5D) or a control vector or treated with 50 U/ml of IFN-β. Sixteen hours later, the cells were washed, infected with HIV-1LAI at an MOI of 1 for 3 h, and further washed, and fresh medium was added. Forty-eight hours later, culture supernatant was collected and infectious HIV-1 production was determined by virus titration assay (n = 3). *, P < 0.001. hpi, hours postinfection; _t_1/2, half-life; NA, not applicable. n, number of samples.

FIG. 3.

IRF-3 depletion requires HIV-1 replication but occurs independently of viral protease function. (A) SupT1 cells were pretreated with 5 μM AZT (Low AZT) or 10 μM AZT (High AZT) and were then mock infected or infected with HIV-1LAI at an MOI of 1. Infection was allowed to continue in the presence of the drug, and the cells were harvested at the indicated times postinfection. Cell lysates were analyzed by immunoblotting to determine the levels of IRF-3, HIV-1 p24, and actin. (B) Cells were treated and infected with HIV-1 as described above, except with 100 nM of the HIV-1 protease inhibitor compound indinavir or darunavir. (C) SupT1 cells were infected with a common aliquot of HIV-1LAI (MOI of 1) that had been divided and left untreated or subjected to UV light inactivation for 15 min prior to addition to SupT1 cells. Values represent the percentages of IRF-3 remaining compared to that at time zero of infection. (D) HEK293 cells were transfected with 100 ng of Flag-IRF-3 and 900 ng of HIV-1 Vif, Vpr, control plasmid, or a mixture of both Vif and Vpr (450 ng each). (Top) Immunoblot of IRF-3, Vpr, Vif, and actin levels in the transfected cells. (Bottom) The bars show the levels of SenV-induced IFN-β promoter activity after coexpression of the same HIV-1 virus constructs with the IFN-β promoter luciferase construct and an internal control. SenV-induced IFN-β promoter activity was determined by infecting each culture of cells with 50 hemagglutinating units of SenV and assessing promoter activity 24 h later (n = 3). *, P < 0.01. hpi, hours postinfection. n, number of samples.

FIG. 4.

Disruption of IRF-3-dependent PRR signaling by HIV-1. (A) poly(I:C) or tRNA was transfected into SupT1 cells that were mock infected or infected with HIV-1LAI (MOI of 1) for 24 h prior to transfection. Cells were harvested at the indicated times posttransfection and subjected to immunoblot analysis of ISG56, IRF-3, HIV p24, and actin protein levels. Values represent the percentages of IRF-3 compared to that at time zero of infection. Data shown are representative of three independent experiments. (B to F) Cells were cotransfected with HIV-1pNL4-3 provirus DNA or control plasmid and the indicated promoter-luciferase plasmid construct. Forty-eight hours posttransfection, the cells were infected with 50 hemagglutinating units/ml of SenV or treated as indicated to induce the indicated PRR signaling, and promoter-luciferase activity was measured 18 h later (n ≥ 3). (B) SenV-induced IFN-β promoter activity in HEK293 cells. *, P < 0.005. (C) SenV-induced ISG56 promoter activity in HEK293 cells. *, P < 0.02. (D) HEK293-TLR3 cells were transfected as described for panel B. Forty-eight hours posttransfection, the cells were treated with 100 μg/ml poly(I:C) added to the culture medium, and IFN-β promoter activity was measured 18 h later. *, P < 0.005. (E) SenV-induced NF-κB promoter activity in HEK293 cells. (F) IFN-β-induced ISRE promoter activity in HEK293 cells. hpt, hours posttransfection; pI:C, poly(I:C). n, number of samples.

FIG. 5.

HIV-1 depletion of IRF-3 compromises innate antiviral immunity to enhance cellular permissiveness for secondary virus infection. (A) HeLa CD4+ cells were mock infected or treated with HIV-1LAI (MOI of 10) for 30 h before the addition (+) of 50 hemagglutinating units/ml of SenV. Eighteen hours later, cells were immunostained with antibodies against IRF-3 and HIV p24. Cell nuclei were stained with DAPI. Cells were visualized by fluorescence microscopy. Merged-color images are shown. (B) SupT1 cells were cultured alone or were infected with HIV-1LAI. Twenty-four hours later, the cells were mock treated or were infected with SenV. Cells were harvested at the indicated times (hours) after SenV infection (hp SenV infection), and total RNA was isolated for qPCR analysis of IFN-β mRNA levels (n = 3). Cells were treated in parallel, and SenV protein and actin levels were analyzed by immunoblotting. Values represent the percentages of SenV protein compared to input. (C) PBMCs were treated as described for panels A and B but were mock infected or infected with HIV-1 for 48 h prior to SenV infection. Cells were harvested and subjected to immunoblot analysis for determination of protein abundance. Data are representative of three independent experiments. n, number of samples.

FIG. 6.

HIV-1 depletion of IRF-3 ex vivo and in vivo. (A) IRF-3, HIV p24, and actin protein levels in vaginal mucosal T cells infected ex vivo with HIV-1BaL or HIV-1JR-CSF (200 ng p24/ml). Virus was added to cultures and allowed to infect for 48 h, after which extracts were prepared and subjected to immunoblot analysis. The relative level of IRF-3 in each culture compared to that for mock-infected cells is presented, and results represent three independent analyses. (B) IRF-3, IRF-7, and actin protein levels in CD4+ or CD4− cells from HIV-1-infected patients. PBMCs from healthy donors or HIV-1 patients were thawed and allowed to rest overnight before the CD4+ and CD4− cells were separated by cell sorting. Extracts from each cell population were analyzed by immunoblotting. Three samples from each group are shown. A total of five samples from seronegative donors (control), five samples from LTNP, and six samples from patients with acute HIV-1 infection were screened. Values represent the percentages of IRF-3 compared to that from the CD4− cell population assessed from the same patient.

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Human immunodeficiency virus type 1 mediates global disruption of innate antiviral signaling and immune defenses within infected cells - PubMed (original) (raw)