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. 2014 Jan 24;343(6169):428-32.
doi: 10.1126/science.1243640. Epub 2013 Dec 19.

IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected with HIV

Kathryn M Monroe  1 , Zhiyuan Yang, Jeffrey R Johnson, Xin Geng, Gilad Doitsh, Nevan J Krogan, Warner C Greene
Affiliations

IFI16 DNA sensor is required for death of lymphoid CD4 T cells abortively infected with HIV

Kathryn M Monroe et al. Science. .

Abstract

The progressive depletion of quiescent "bystander" CD4 T cells, which are nonpermissive to HIV infection, is a principal driver of the acquired immunodeficiency syndrome (AIDS). These cells undergo abortive infection characterized by the cytosolic accumulation of incomplete HIV reverse transcripts. These viral DNAs are sensed by an unidentified host sensor that triggers an innate immune response, leading to caspase-1 activation and pyroptosis. Using unbiased proteomic and targeted biochemical approaches, as well as two independent methods of lentiviral short hairpin RNA-mediated gene knockdown in primary CD4 T cells, we identify interferon-γ-inducible protein 16 (IFI16) as a host DNA sensor required for CD4 T cell death due to abortive HIV infection. These findings provide insights into a key host pathway that plays a central role in CD4 T cell depletion during disease progression to AIDS.

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Figures

Figure 1
Figure 1. Biochemical analysis of cytosolic DNA binding proteins in CD4 T-cells
(A) Tonsillar CD4 T-cell lysates were incubated with a 500-bp biotinylated HIV Nef DNA probe or control non-biotinylated DNA and immunoprecipitated with streptavidin-coated beads. Samples were separated by SDS-PAGE and silver stained. (B) Western blot analysis of nuclear histone H3 and beta-actin in whole or digitonin lysis buffer prepared CD4 T-cell lysates. (C) Top ranked hits (rank based on protein discriminant scores described in Materials and Methods) from MS samples prepared as in (A). (D) Western blot analysis of candidate DNA sensors. (E) SDS-PAGE and silver stain analysis of biotinylated dsDNA or ssDNA samples prepared as in (A) and competed with a 10-fold excess of ssDNA or dsDNA (F) Western blot analysis of IFI16 and RIG-I binding samples in (E). (G) Western blots with high levels of protein input showing IFI16 binding to biotinylated ssDNA and dsDNA and RIG-I-RNA controls.
Figure 2
Figure 2. IFI16 shRNA knockdown rescues activated and rested splenic CD4 T-cells from depletion following abortive HIV infection
(A) Western blot analysis of IFI16 and beta-actin expression in shRNA expressing mCherry+ CD4+ T-cells after activation and rest in reduced IL-2. (B) Flow cytometry analysis of mCherry-positive CD4 T-cell survival after knockdown with shSCR or shIFI16-A and co-culture with either donor-matched mCherry− CD4+ T-cells or tonsillar HLAC spinoculated with an HIV-1-GFP reporter virus. Cells were co-cultured in the presence or absence of 100 nM efavirenz, or with uninfected cells. Data represent the average of three independent experiments from three different donors. Error bars indicate standard error of the mean, * p<0.05 (Student’s t-test), n.s.=not significant, p>0.1. (C) Flow cytometry analysis of CD25 and CD69 expression after IL-2 reduction. (D) Flow cytometry analysis of mCherry+ GFP+ populations in shRNA-expressing spleen cells post-co-culture.
Figure 3
Figure 3. shRNA knockdown of IFI16 rescues HIV-induced tonsillar CD4 T-cell depletion
(A) Western blot analysis of IFI16 and beta-actin expression in mCherry+ tonsil CD4 T cells receiving shScramble, shIFI16-A or shIFI16-B. (B) Quantitation of flow cytometry of HLAC infected with VLP-Vpx, followed by shScramble, shIFI16-A, B lentiviruses pseudotyped with HIV gp160 Env then co-cultured with 293T cells producing HIV-1. ** p≥0.01 (Student’s t-test), n.s.=not significant, p>0.1. (C) Western blot analysis of shIFI16-C knockdown. (D) Quantitation of flow cytometry results as in (B). *** p<0.001. (E) Quantitation of mCherry+ gate of HLAC treated as in (B) with single round HIV-1ΔEnv pseudotyped with gp160 envelope or HIV-1 D116N integrase mutant. ** p<0.01, * p<0.05. (F) Flow cytometry analysis of FLICA-660 Caspase-1 and IFNβ intracellular staining in mCherry+ cells, histograms are representative of results obtained with two donors.
Figure 4
Figure 4. VLP-Vpx-facilitated shRNA knockdown of other candidate DNA sensors does not rescue cells from depletion following abortive HIV infection
(A) Western blot analysis of AIM2, STING, and HSP90 in shRNA expressing mCherry+ THP-1 cells. (B) Quantitation of flow cytometry results for HLAC infected with shScramble, shAIM2, or shSTING. n.s.=not significant, p>0.1 (Student’s t-test). (C) Western blot analysis of IFIX in mCherry+ SupT1 cells. (D) Quantitation of flow cytometry analysis as in (B) of shScramble and shIFIX. (E) Western blot analysis of DNPK-1 in shRNA expressing mCherry+ Jurkat T-cells. (F) Flow cytometry analysis as in (B) with shDNPK-1. (G) CFSE labeled HLAC were pre-treated with DMSO alone in uninfected and no drug conditions, 10 or 20 μM Nu7026 or 1 or 2 μM Nu7441 or 250 nM AMD3100. CFSE+ cells were co-cultured with donor-matched HLAC productively infected with HIV and analyzed 3 days post co-culture. Quantified data represent the average of three independent experiments from three different donors. Error bars represent the standard errors of the mean. (H) Summary model.
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