1. Volume 20, Issue 4, Pages 443-457 (October 2016)
cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites 
Shuting Xu, Aurélie Ducroux, Aparna Ponnurangam, Gabrielle Vieyres, Sergej Franz, Mathias Müsken, Thomas Zillinger, Angelina Malassa, Ellen Ewald, Veit Hornung, Winfried Barchet, Susanne Häussler, Thomas Pietschmann, Christine Goffinet 
Cell Host & Microbe 
Volume 20, Issue 4, Pages (October 2016)
DOI: /j.chom
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2. Cell Host & Microbe 2016 20, 443-457DOI: (10.1016/j.chom.2016.09.003)
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 1
Coculture of Env-Expressing cGAS-Positive Donor Cells and Primary Human Macrophages Induces Type I IFN
(A) Indicated CHO cells were cocultured with macrophages in the presence or absence of T-20. Induction of human Ifn-β mRNA expression and secretion of bioactive IFN are shown. Error bars indicate SEM from five experiments.
(B) Induction of human Ifn-β mRNA expression in macrophages cocultured with indicated small interfering RNA (siRNA)-transfected CHO cells is shown. Data were obtained from one experiment representative out of five. Significance was calculated using two-tailed Student’s t test. Supernatants were probed for bioactive IFN. Δ < 1.5 IU/mL. Shown are results from one experiment representative out of two.
(C) Shown are induction of human Ifn-β mRNA expression and release of bioactive IFN in macrophages cocultured with indicated CHO or Jurkat cells. Data were obtained from one experiment representative out of three.
(D) Indicated Jurkat cells expressing cGAS-GFP variants were cocultured with macrophages. Shown is release of bioactive IFN. Data were obtained from one experiment representative out of three. Lysates of indicated Jurkat cells were analyzed for expression of indicated proteins by immunoblotting.
(E) Shown is release of bioactive IFN from macrophages transfected with small molecules extracted from indicated cell lines or pure cGAMP. Shown are results from one experiment representative out of two.
(F) Macrophages were transfected with SVPDE-treated or control-treated cGAMP or CHO-derived small molecules. Shown is release of bioactive IFN. Shown are results from one experiment representative out of three.
Error bars indicate, if not otherwise stated, SD obtained from one representative experiment out of two.
See also Figures S1–S4.
Cell Host & Microbe  , DOI: ( /j.chom )
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4. Figure 2
Membrane Fusion-Induced Type I IFN Response Is STING-TBK-1 Dependent
(A) Shown is the relative expression of cGAS and STING in macrophages after transfection of specific siRNAs. Error bars represent SEM from three independent experiments. Significance was calculated using two-tailed Student’s t test.
(B) siRNA-transfected macrophages were cocultured with indicated CHO cells in the presence or absence of T-20. Induction of human Ifn-β mRNA expression and release of bioactive IFN upon coculture are shown. Dotted line, assay background. Data were obtained from one experiment representative out of two and three, respectively.
(C) Shown is induction of human Ifit1 mRNA expression in indicated THP-1 cells upon coculture with indicated CHO cells. Expression of STING and cGAS is analyzed by immunoblotting.
(D) Primary macrophages were pretreated with DMSO or BX795 for 48 hr, followed by coculture with indicated CHO cells. Induction of human Ifn-β mRNA expression and release of bioactive IFN upon coculture are shown. Data were obtained from one experiment representative out of three.
Error bars indicate, if not otherwise stated, SD obtained from one representative experiment out of two.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 3
Mounting of Coculture-Induced Type I IFN Requires HIV-1 Env and CD4/Coreceptor-Mediated Membrane Fusion and Direct Cell-Cell Contacts
(A) Indicated CHO cells were cocultured with primary macrophages in the presence or absence of T-20, C34, AMD3100, DMSO, or Maraviroc. Hybrids were assessed for induction of human Ifn-β mRNA expression. Supernatants were probed for bioactive IFN. Error bars indicate SEM from three to five experiments.
(B) Indicated CHO cells were cocultured with primary macrophages in the presence or absence of control IgG, anti-CD4, or anti-gp120 antibodies 2G12, , and 3BNC117. Supernatants were probed for bioactive IFN. Δ < 1.5 IU/mL.
(C) CHO cells expressing indicated HIV-1 Env variants or empty vector were cocultured with primary macrophages in the presence or absence of T-20. Hybrids were assessed for induction of human Ifn-β mRNA expression. Supernatants were probed for release of bioactive IFN. Dotted line, assay background. Data were obtained from one experiment representative out of two.
(D) Jurkat cells expressing indicated HIV-1 Env variants were cocultured with primary macrophages in the presence or absence of T-20. Supernatants were probed for bioactive IFN. Error bars indicate SEM from five experiments.
(E) Primary macrophages and CHO cells were cocultured either directly or in transwells. Relative levels of human Ifn-β and Ifit1 mRNA expression are shown. Supernatants were probed for bioactive IFN. Δ < 1.5 IU/mL. Data were obtained from one experiment representative out of two.
Error bars indicate, if not otherwise stated, SD obtained from one representative experiment out of two.
See also Figure S2.
Cell Host & Microbe  , DOI: ( /j.chom )
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6. Figure 4
Induction of Type I IFN Involves T-20-Sensitive Microfusion Events between T Cells and Primary Macrophages
(A) Indicated Jurkat cells and macrophages were stained with indicated dyes, respectively, prior to coculture in absence or presence of T-20 or Jasplakinolide (0.5 μM). Shown are representative dot plots from one out of six to ten experiments. Numbers in P2 indicate percentages of green events within the respective living gate P1.
(B) Quantitative analysis of green dye acquisition by red CMTPX-positive macrophages after coculture with green CMFDA-positive T cells. Percentages obtained for association of Jurkat-Env T cells with macrophages in absence of treatment were set to 100%. Error bars indicate SEM of three to ten experiments. Significance was calculated using two-tailed Student’s t test.
(C) cGAS-expressing Jurkat-Env cells were cocultured with macrophages in the presence of DMSO or Jasplakinolide (0.5 μM). Shown is the relative release of bioactive IFN upon coculture. Error bars indicate SEM from three experiments.
(D) Time-lapse sequence of green CMFDA-labeled Jurkat-Env and red CMTPX-labeled macrophages in presence or absence of T-20. Imaging starts at 3 hr post-coculture initiation. Asterisks indicate sites of cell-cell interactions and intercellular transfer of cell tracker dye. Scale bar, 10 μm.
See also Figure S5 and Movies S1 and S2.
Cell Host & Microbe  , DOI: ( /j.chom )
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7. Figure 5
Coculture-Induced Type I IFN Results in a Functional Antiviral State and Protects from HIV-1 Infection
(A) Indicated CHO cells were cocultured with primary human macrophages in the presence or absence of T-20 or AMD3100. Hybrids were assessed for induction of indicated mRNA expression. Data were obtained from one experiment representative out of two to three.
(B) Primary macrophages were pretreated with supernatant of indicated cocultures prior to infection with HIV-1Ba-L. Infection efficiency was monitored by FACS-based analysis of intracellular p24CA immunostaining. Numbers indicate the concentration of IFN (in IU/mL) measured in the coculture supernatant used for the inoculation of naive macrophages prior to infection. Macrophages of donor 3 were also treated with pure IFN-α2a (50 IU/mL).
(C) Concentration of released bioactive IFN and the inhibition of HIV-1 infection are plotted against each other. For these values, the Pearson’s correlation coefficient R and the corresponding p value are shown. The open circle depicts the value obtained for pure IFN-α2a.
(D) After coculture with indicated Jurkat cells, primary macrophages were challenged with HIV-1Ba-L. Infection efficiency was monitored by FACS-based analysis of intracellular p24CA immunostaining. T-20 was applied during coculture and omitted during HIV-1 challenge. Note: potentially remaining Jurkat cells were refractory to HIV-1 infection due to absent CD4 surface expression.
Cell Host & Microbe  , DOI: ( /j.chom )
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8. Figure 6
HIV-1-Infected PBLs, but Not Cell-Free Virions, Convey a Type I IFN Response in Cocultured Autologous Macrophages
(A) Relative cGAS mRNA and protein expression were analyzed in uninfected and HIV-1-infected PBLs. cGAS mRNA expression in THP-1 was set to 100%. Numbers indicate the percentage of p24CA-positive cells within the respective cultures.
(B) Macrophages were infected with cell-free virus or cocultured with HIV-1-infected autologous PBLs in the presence or absence of T-20. HIV-1 infection of macrophages was monitored over time by FACS-based analysis of intracellular p24CA immunostaining.
(C) Supernatants of the experiment shown in (B) and of the separate PBL monoculture were probed for bioactive IFN release using the HL116-based IFN luciferase reporter system.
(D and E) Macrophages were infected with cell-free virus (D) or cocultured with HIV-1-infected or uninfected autologous PBLs (D and E) in the presence or absence of indicated drugs and antibodies. HIV-1 infection of macrophages was monitored over time by FACS-based analysis of intracellular p24CA immunostaining. Supernatants were probed for bioactive IFN release using the HL116-based IFN luciferase reporter system. Shown are the results of two different cocultures.
(F) Same set-up like (B) and (C). Infected PBLs and macrophages were separated by transwells (TW) where indicated.
See also Figures S2, S6, and S7.
Cell Host & Microbe  , DOI: ( /j.chom )
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9. Figure 7
The Type I IFN Response that Is Induced in Primary Macrophages upon Interaction with HIV-1-Infected PBLs Coincides with cGAMP Presence in PBLs and Requires Functional STING in Target Macrophages
(A) Macrophages were transfected with SVPDE-treated or control-treated small molecules extracted from sucrose-purified virions and corresponding producer PBLs. Supernatants of transfected macrophages were probed for bioactive IFN. Numbers indicate the percentage of p24CA-positive cells within the respective infected PBL cultures from which small molecules were extracted. Western blots show the amount of p24CA in the respective cell-free and cell-associated material used for small-molecule extraction.
(B) cGAS and STING mRNA expression levels were reduced by transfection of macrophages with specific siRNAs. Shown is the relative expression of cGAS and STING obtained for (C) and (D).
(C) Indicated macrophages were cocultured with autologous HIV-1-infected PBLs or challenged with cell-free virions. Supernatants were probed for bioactive IFN.
(D) HIV-1 infection of macrophages was monitored by FACS-based analysis of intracellular HIV-1 p24CA immunostaining. Shown is one experiment representative out of three.
Error bars indicate, if not otherwise stated, SD obtained from one representative experiment out of two.
See also Figure S7.
Cell Host & Microbe  , DOI: ( /j.chom )
Copyright © 2016 Elsevier Inc. Terms and Conditions