IFI44 suppresses HIV-1 LTR promoter activity and facilitates its latency - PubMed (original) (raw)

IFI44 suppresses HIV-1 LTR promoter activity and facilitates its latency

Derek Power et al. Virology. 2015 Jul.

Abstract

IFI44 is an interferon-alfa inducible protein, and is associated with infection of several viruses. However, IFI44 elicits minimal antiviral effects on these viruses, and its exact role is still unknown. Here we show that IFI44 inhibits HIV-1 replication in vitro. Through depletion of endogenous IFI44 or overexpression of IFI44 we confirm that IFI44 suppresses HIV-1 LTR promoter activity and affects viral transcription. Furthermore, we find that IFI44 localizes to nuclei and binds to the HIV-1 LTR promoter in HIV-1 infected cells. Removing suppression of HIV-1 transcription benefits reactivation of HIV-1 proviruses for purging latent reservoirs. We demonstrate that depletion of endogenous IFI44 in J-LAT cells induces reactivation of latent HIV-1. Based on these results, we propose a model in which IFI44 is recruited to the HIV-1 LTR, which may suppress viral transcription and prevent reactivation of latent HIV-1. Our study suggests a previously unrecognized anti-HIV phenomenon for interferon-stimulated proteins.

Keywords: HIV-1; IFI44; ISG; Interferon; LTR; Latency; Reactivation; Transcription.

Copyright © 2015 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

IFI44 depletion by RNAi increases HIV-1 infection. (A). MAGI-HeLa was transfected with sequence-unique IFI44 siRNA, siIFI44-1 or siIFI44-2, or non-targeting control siRNA (siNT). 72 hours post-transfection, total RNA was extracted for reverse transcription and quantitative real-time PCR to measure the IFI44 mRNA level. Data were normalized to the siNT-treated cells. (B). MAGI-HeLa was transfected with siIFI44-1, siIFI44-2, or non-targeting siNT. 48 hours post-transfection, cells were treated with IFN-α, or mock treated for 24 hours. Cells were then infected with HIV-IIIB wild-type viruses for 48 hours, and stained with anti-HIV-1 p24 CA antibody (anti-CA) and a FITC anti-mouse secondary antibody. Nuclei were stained using Hoechst. The percentage of infected cells was measured and normalized to the siNT-treated cells. (C, D). MAGI-HeLa was transfected with siIFI44-1, siIFI44-2, or siNT. 72 hours post-transfection, cells were infected with VSV-G pseudo-typed (C) HIV-NL4-3-GFP [Δ Env] or MLV-GFP virus [Δ Env], or (D) LTR-GFP or CMV-ZSG lentivirus for 48 hours. Cells were stained with Hoechst and the percentage of GFP-positive cells was measured and normalized to the siNT-treated cells. (E). Sequence-unique IFI44 shRNA, shIFI44-1, shIFI44-2, or control firefly luciferase shRNA (shFLuc) in pAPM vector was transduced in Jurkat T cells. Cells were subjected to RNA extraction, reverse transcription, and quantitative real-time PCR for measuring the IFI44 mRNA level. Data were normalized to the shFLuc-transduced cells. (F). Jurkat T cells stably expressing shIFI44-1, shIFI44-2, or shFLuc were infected with VSV-G pseudo-typed HIV-NL4-3-GFP [Δ Env] viruses for 48 hours. Cells were assessed using flow cytometry and the percentage of GFP-positive cells were calculated and normalized to the shFLuc-transduced cells. All results were presented as mean ± s.d. (n = 3); * P < 0.05 from _t_-test.

Figure 2

Figure 2

Exogenous expression of IFI44 suppresses HIV-1 viral gene expression. (A). pQCXIP-HA-IFI44 or pQCXIP empty vector was transduced into MAGI-HeLa cells. Cell lysate was prepared and analyzed for HA-IFI44 expression by western blot using an anti-HA antibody. (B). MAGI-HeLa cells stably expressing HA-IFI44 or empty vector were pretreated with IFN-α or mock-treated for 24 hours. Cells were infected with HIV-IIIB wild-type viruses for 48 hours and stained with an anti-HIV-1 p24 CA antibody (anti-CA) and Hoechst. The percentage of infected cells was measured and normalized to the vector-only cells. (C). pQCXIP-HA-IFI44 or pQCXIP empty vector was transduced into HEK293 cells. Cell lysate was prepared and analyzed for HA-IFI44 expression by western blot using an anti-HA antibody. (D). HEK293 cells stably expressing HA-IFI44 or empty vector were co-transfected with HIV-LTR firefly luciferase reporter and pRL-TK renilla luciferase control vectors, together with pcDNA-FLAG-TAT vector. Luciferase activity was measured 24 hours post-transfection and normalized to the empty vector cells. (E). pQCXIP-HA-IFI44 or pQCXIP empty vector was transduced into JLTRG cells. Total RNA was extracted for reverse transcription and quantitative real-time PCR to measure the IFI44 mRNA level. Data were normalized to the JLTRG-Vector cells. (F). JLTRG cells stably expressing HA-IFI44 or empty vector were transiently transfected with pcDNA-FLAG-TAT. 48 hours post-transfection, cells were assessed by flow cytometry, and the percentage of GFP-positive cells was normalized to the vector-only cells. All results were presented as mean ± s.d. (n = 3); * P < 0.05 from _t_-test.

Figure 3

Figure 3

IFI44 enters the nuclei and associates with the HIV-1 LTR upon HIV-1 infection. (A). HEK293 cells stably expressing HA-IFI44 were infected with VSV-G pseudo-typed HIV-NL4-3-GFP [Δ Env] viruses (+HIV, MOI=5) or mock treated (−HIV) for 48 hours. Equal numbers of cells were subjected to cytoplasmic (cyt) and nuclear (nuc) fractionation. Extracted protein samples were separated by SDS-PAGE. HA-IFI44 cyt or nuc localization was analyzed by western blot using an anti-HA antibody. GAPDH protein level in cyt or nuc extraction was determined to indicate equal loading. E-cadherin (cytoplasm) and JMJD1A (nuclei) were stained to ensure the separation of cytoplasmic and nuclear proteins. (B). HEK293 cells stably expressing HA-IFI44 were infected with VSV-G pseudo-typed HIV-1 NL4-3-GFP [Δ Env] viruses. Cells were subjected to ChIP assays using an anti-HA antibody or a rabbit control IgG. Precipitated DNA samples were extracted and analyzed by PCR using primers amplifying the nuc-0, L1 nucleosome-free region (PPR), nuc-1, and nuc-2 regions of the HIV-1 LTR promoter.

Figure 4

Figure 4

IFI44 depletion reverses HIV-1 latency in J-LAT A2 cells. (A). shIFI44-1, shIFI44-2, or shFLuc in pAPM vector was transduced into J-LAT A2 cells. Cells were subjected to RNA extraction, reverse transcription, and quantitative real-time PCR for measuring the IFI44 mRNA level. Data were normalized to the shFLuc-transduced cells. (B). Equal numbers of J-LAT A2 cells stably expressing shIFI44-1, shIFI44-2, or shFLuc were assessed by flow cytometry, and the percentage of GFP positive cells (bottom right quadrant) were measured. Data are one representative of three independent experiments. (C). Percentage of GFP positive cells from three independent flow cytometry assays for J-LAT A2 cells expressing shIFI44-1 or shIFI44-2 was averaged and normalized to that of J-LAT A2 cells expressing shFLuc. All results were presented as mean ± s.d. (n = 3); * P < 0.05 from _t_-test.

References

    1. Baca-Regen L, et al. Alpha interferon-induced antiretroviral activities: restriction of viral nucleic acid synthesis and progeny virion production in human immunodeficiency virus type 1-infected monocytes. J Virol. 1994;68(11):7559–65. -PMC -PubMed
    1. Coccia EM, Krust B, Hovanessian AG. Specific inhibition of viral protein synthesis in HIV-infected cells in response to interferon treatment. J Biol Chem. 1994;269(37):23087–94. -PubMed
    1. Fernie BF, Poli G, Fauci AS. Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T-lymphocytic cells. J Virol. 1991;65(7):3968–71. -PMC -PubMed
    1. Gendelman HE, et al. Regulation of HIV replication in infected monocytes by IFN-alpha. Mechanisms for viral restriction. J Immunol. 1990;145(8):2669–76. -PubMed
    1. Gendelman HE, et al. Restriction of HIV replication in infected T cells and monocytes by interferon-alpha. AIDS Res Hum Retroviruses. 1990;6(8):1045–9. -PubMed

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