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. 2015 Jul;89(13):6575-84.
doi: 10.1128/JVI.00889-15. Epub 2015 Apr 8.

HIV Blocks Interferon Induction in Human Dendritic Cells and Macrophages by Dysregulation of TBK1

Andrew N Harman  1 , Najla Nasr  1 , Alexandra Feetham  1 , Ani Galoyan  1 , Abdullateef A Alshehri  1 , Dharshini Rambukwelle  1 , Rachel A Botting  1 , Bonnie M Hiener  1 , Eve Diefenbach  1 , Russell J Diefenbach  1 , Min Kim  1 , Ashley Mansell  2 , Anthony L Cunningham  3
Affiliations

HIV Blocks Interferon Induction in Human Dendritic Cells and Macrophages by Dysregulation of TBK1

Andrew N Harman et al. J Virol. 2015 Jul.

Abstract

Dendritic cells (DCs) and macrophages are present in the tissues of the anogenital tract, where HIV-1 transmission occurs in almost all cases. These cells are both target cells for HIV-1 and represent the first opportunity for the virus to interfere with innate recognition. Previously we have shown that both cell types fail to produce type I interferons (IFNs) in response to HIV-1 but that, unlike T cells, the virus does not block IFN induction by targeting IFN regulatory factor 3 (IRF3) for cellular degradation. Thus, either HIV-1 inhibits IFN induction by an alternate mechanism or, less likely, these cells fail to sense HIV-1. Here we show that HIV-1 (but not herpes simplex virus 2 [HSV-2] or Sendai virus)-exposed DCs and macrophages fail to induce the expression of all known type I and III IFN genes. These cells do sense the virus, and pattern recognition receptor (PRR)-induced signaling pathways are triggered. The precise stage in the IFN-inducing signaling pathway that HIV-1 targets to block IFN induction was identified; phosphorylation but not K63 polyubiquitination of TANK-binding kinase 1 (TBK1) was completely inhibited. Two HIV-1 accessory proteins, Vpr and Vif, were shown to bind to TBK1, and their individual deletion partly restored IFN-β expression. Thus, the inhibition of TBK1 autophosphorylation by binding of these proteins appears to be the principal mechanism by which HIV-1 blocks type I and III IFN induction in myeloid cells.

Importance: Dendritic cells (DCs) and macrophages are key HIV target cells. Therefore, definition of how HIV impairs innate immune responses to initially establish infection is essential to design preventative interventions, especially by restoring initial interferon production. Here we demonstrate how HIV-1 blocks interferon induction by inhibiting the function of a key kinase in the interferon signaling pathway, TBK1, via two different viral accessory proteins. Other viral proteins have been shown to target the general effects of TBK1, but this precise targeting between ubiquitination and phosphorylation of TBK1 is novel.

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Figures

FIG 1
FIG 1
Human dendritic cells and macrophages do not produce type I or type III interferons in response to HIV-1 due to a failure of IRF3 activation. MDDCs and MDMs were treated with purified HIV-1BaL at an MOI of 3 (MDDCs) or 1 (MDMs) for 2 to 96 h or with HSV-2186 at an MOI of 1 or Sendai virusCantell (SV) for 2 to 24 h. (A) Type I and III IFN gene expression was determined by qPCR, and data for Sendai virus and HSV-1 at 18 h only are shown as a positive control for IFN induction. Data were normalized to GAPDH expression and are represented as log2 fold change in the ratio of mean treatment values to mean mock control values (mean ± standard error of the mean [SEM], n = 5) (P values were calculated using a paired two-tailed t test: *, P < 0.05; **, P < 0.01). No upregulation of IFN-κ, -λ2, or -λ3 mRNA was detected in either cell type in response to any treatment. (B) Nuclear translocation of IRF3 in MDDCs was visualized by immunofluorescence microscopy, with IRF3 shown in green and the nucleus (DAPI staining) shown in blue. Images were taken using a DeltaVision deconvolution microscope at a magnification of ×60. Representative images from four experiments at 14 h after virus treatment are shown. (C) Phospho-IRF3 and GAPDH protein expression levels in MDDCs were determined by Western blotting. Representative data from five independent experiments are shown.
FIG 2
FIG 2
HIV-1 is sensed by human dendritic cells and macrophages, resulting in the recruitment of TBK1 signaling complexes associated with type I interferon induction. (A) Day 6 MDDCs and MDMs were treated with LPS (10 μg/ml) for 2 h or HIV-1BaL (MOI of 1) for 6 h or mock treated, and the TBK1 distribution was determined by immunofluorescence deconvolution microscopy at a magnification of ×60. TBK1 is shown in green and the nucleus (DAPI staining) in blue. (B and C) Day 6 MDDCs were treated with HIV-1BaL or Sendai virus or mock treated for 6 h, and proximity ligation assays (PLA) were carried out for the following pairs of antibodies: mouse anti-TBK1 and rabbit anti-TRAF3, rabbit anti-TBK1 and mouse anti-IRF3, and rabbit anti-TRAF3 and mouse anti-IRF3. After nucleic acid ligation, rolling-circle amplification, and incubation with an FITC-conjugated HIV-1 p24 antibody and DAPI, red fluorescent dots, indicative of an interaction between two proteins of interest, were counted. Green dots are HIV-1 particles. (B) Representative images are shown for TBK1/TRAF3, TRAF3/IRF3, and TBK1/IRF3. (C) The graphs represent quantitative analysis of the number of PLA fluorescent red puncta counted within a mean of 100 cell profiles ± one SE. Positive cells counted are representative of three donors. Outlier cells containing large numbers of puncta are shown with an X. P values were calculated using a paired two-tailed t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) MDDCs were treated with HIV-1BaL (MOI of 1) or Sendai virus or mock treated for 4 h. A coimmunoprecipitation assay was then carried out using an antibody to TBK1. Western blotting was then conducted on the immunoprecipitated TBK1 complex and the blots probed for TRAF3 (upper band) and TBK1 (lower band).
FIG 3
FIG 3
Interferon-inducing signal transduction proteins are not degraded in HIV-1-treated dendritic cells and macrophages. MDDCs (A) and MDMs (B) were mock treated or treated with HIV-1BaL at an MOI of 1 (MDMs) or 3 (MDDCs) for 6 to 96 h, and the expression levels of TBK1, TRAF3, IRF3, and STING were determined by Western blotting (results are representative of three experiments).
FIG 4
FIG 4
TBK1 is ubiquitinated but not phosphorylated in HIV-1-treated MDDCs and MDMs. (A) TBK1 and IKKε levels were determined in increasing amounts (15, 30, and 60 μg) of mock MDDC cell lysates. The IKKε image was acquired with a much longer exposure time than that for TBK1 in order to visualize the very faint bands (results are representative of three experiments). (B and C) MDDCs and MDMs were mock treated or treated with HIV-1BaL at an MOI of 1 (MDMs) or 3 (MDDCs) for 6 to 96 h or with Sendai virus for 4 to 24 h (results are representative of five experiments). (B) A TBK1 pulldown assay was conducted on all cell lysates and K63 ubiquitination and TBK1 expression levels determined by Western blotting using anti-K63 ubiquitin and anti-TBK1 antibodies. (C) Phospho-TBK1 expression levels were determined by Western blotting using a phospho-TBK1 antibody.
FIG 5
FIG 5
Defining HIV-1 accessory proteins that bind TBK1. (A) Day 2 MDDCs were treated with VSV-G-pseudotyped HIV-1 accessory protein deletion mutants for 24 h and IFN-β gene expression levels determined by qPCR. Data were normalized to GAPDH expression and are represented as the log2 fold change of the ratio of mean HIV-1 treatment values to mean mock control values (mean ± SEM, n = 5) (P values were calculated using a paired two-tailed t test: *, P < 0.05 **). (B and C) HEK293T cells were mock transfected or transfected with mammalian expression vectors encoding TBK1 or Vpr-GFP (B) or TBK1 or HIV-1BaL provirus (pBaL) (C). The TBK1 and Vpr-GFP (B) or TBK1 and pBaL (C) cell lysates were then mixed, and a coimmunoprecipitation assay was then carried out using an antibody to TBK1, which was left overnight. Western blotting was then conducted on the immunoprecipitated TBK1 complex, and the blots probed for TBK1 and Vpr (B) and TBK1, Vpr, Vif, and Nef (C). The data presented are representative of three experiments.
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