The cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency virus type 1

Abstract

Viral infection triggers innate immune sensors to produce type I interferon. However, infection of T cells and macrophages with human immunodeficiency virus (HIV) does not trip those alarms. How HIV avoids activating nucleic acid sensors is unknown. Here we found that the cytosolic exonuclease TREX1 suppressed interferon triggered by HIV. In Trex1(-/-) mouse cells and human CD4(+) T cells and macrophages in which TREX1 was inhibited by RNA-mediated interference, cytosolic HIV DNA accumulated and HIV infection induced type I interferon that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate interferon expression via a pathway dependent on the kinase TBK1, the adaptor STING and the transcription factor IRF3. HIV-stimulated interferon production in cells deficient in TREX1 did not involve known nucleic acid sensors.

Fig. 1. TREX1 deficiency inhibits HIV replication and activates IFN-β in response to HIV infection

WT or Trex1^−/− primary MEFs were infected with VSV-G-pseudotyped single round HIV virus. (a) HIV infection, measured by Luc activity 48 hpi, is reduced in Trex1^−/− cells. Luc reporter expression was driven by the HIV LTR in the context of a near full length viral genome (ΔEnv, Luc replacing Nef). (b-d) Cytokine induction, measured by qRT-PCR (b, c) and ELISA (d) 22 hpi, is inhibited by RT, but not IN, inhibitors. (e,f) RT and IN inhibitors both reduce HIV-Luc replication 48 hpi, but only the RT inhibitor suppresses late reverse transcripts (late RT) measured 10 hpi. Both inhibitors were added at the same time as the virus. (g) Virus-like particles (VLP) and heat-inactivated HIV (95°C, 5 min) do not induce substantial IFN-β in either WT or Trex1^−/− cells. Equivalent amounts of HIV (heat inactivated or not) or VLP were used for infection based on p24 ELISA measurements. (h) HIV autointegration is reduced by inhibiting either RT or IN. Error bars indicate S.D. of at least three independent experiments. Data from WT MEF are indicated by black bars; from Trex1^−/− MEF by white bars.

Fig. 2. HIV-stimulated IFN expression is IRF3-dependent

Wild type (WT), Trex1^−/− (KO) or Trex1^−/−Irf3^−/− (DKO) primary MEFs were infected as in Fig. 1. (a) HIV-stimulated IFN-β induction, assessed by qRT-PCR 22 hpi, is eliminated in the absence of IRF3. (b) Irf3-deficiency partially rescues HIV infection in Trex1-deficient cells. HIV infection was measured by Luc activity 48 hpi. (c) Increased HIV autointegration in Trex1-deficient cells is not affected by Irf3-deficiency. Error bars indicate S.D. of at least three independent experiments. (d) IRF3 translocates to the nucleus in HIV-infected Trex1^−/− cells. WT and Trex1^−/− MEFs that were either uninfected or infected with HIV were stained 22 hpi for IRF3 (red) and DAPI (blue). (e) Expression of GFP from an HIV reporter virus (HIV-GFP) is reduced in Trex1^−/− MEF compared to WT MEF. Flow cytometry analysis of GFP expression was measured 24 hpi. A representative plot from 3 independent experiments is shown.

Fig. 3. Cytosolic HIV DNA in Trex1^−/− cells is the trigger for IFN expression

(a) Cytosolic HIV DNA accumulates at higher levels early after infection in Trex1^−/− (KO) than in WT primary MEFs. IFN-β expression lags behind DNA build up and only occurs in Trex1^−/− cells. HIV DNA and IFN-β mRNA were measured by qPCR and qRT-PCR, respectively. (b) Trex1 deficiency does not affect viral entry. Comparable numbers of virions enter WT and Trex1^−/− cells as determined by qRT-PCR of HIV genomic RNA (gRNA) assessed 2 and 5 hpi. (c-e) Increasing multiplicity of infection (MOI) leads to more cytosolic HIV DNA accumulation and IFN-β expression in Trex1^−/− MEF, but not to more chromosomal integration, compared to WT MEF. WT and Trex1^−/− MEFs were infected with HIV at indicated MOI. IFN-β mRNA (c) and cytosolic HIV DNA (d) were measured as in (a). Integrated DNA (e) was measured by a two-step semi-quantitive PCR assay (, see ONLINE METHODS). (f) Conditioned medium from Trex1^−/− MEF, but not from WT and Trex1^−/−Irf3^−/− (DKO) MEF, inhibits de novo HIV infection. Conditioned medium, obtained from cells that were either uninfected or infected with HIV-GFP, was added to WT MEFs that were then infected with HIV-Luc. Luc activity was measured 48 hpi. (g) IFN-β contributes to most of the antiviral activity secreted by Trex1^−/− MEF. Conditioned medium was pre-incubated with or without mouse IFN-β neutralizing antibody before being added to WT MEFs with HIV-Luc. (h) The antiviral effect of varying concentrations of purified recombinant mIFN-β on HIV-Luc infection in WT MEF. *, P <0.01, Student's t-test. Error bars indicate S.D. of three independent experiments.

Fig. 4. Recognition of HIV RT products by enzymatically active TREX1 suppresses IFN induction

(a) Schematic of synthetic nucleic acids used in (b) as they are generated during reverse transcription. (b) DNA-containing RT products trigger a stronger IFN response in Trex1^−/− compared to WT cells. IFN-β expression was measured 6 h post transfection of indicated nucleic acids. (c) TREX1 metabolizes reverse-transcribed HIV DNA and transfected ssDNA and dsDNA in both mouse and human fibroblasts. WT and KO MEFs or WT and TREX1 mutant (D18N) human fibroblasts were either infected with HIV or transfected with ssDNA and dsDNA gag sequence oligonucleotides (100 bp), and cytosolic DNA was measured by qPCR using gag primers 10 hpi or 3 h post transfection. *, P <0.01, Student's t-test. Error bars indicate S.D. of three independent experiments. (d) HIV DNA, but not RNA, is pulled down with FLAG antibody from cytosolic lysates of HeLa-CD4 cells expressing FLAG-TREX1 that were infected with HIV_IIIB for 10 h. Lysates were immunoprecipitated (IP) with FLAG or IgG antibodies. DNA and RNA extracted from the IP were quantified by qPCR or qRT-PCR, respectively. *, P <0.05, Student's t-test. Error bars indicate S.D. of triplicate replicates of two independent experiments. (e,f) TREX1 enzymatic activity is required to support HIV infection. (e) WT and Trex1^−/− (KO) MEFs expressing GFP alone or wild type or enzymatically inactive mutant (D18N) GFP-tagged TREX1 were infected with HIV-Luc, and luciferase activity was measured 48 hpi. TREX1 expression was comparable for each construct by immunoblot. (f) GFP-TREX1, but not the D18N mutant, eliminates excess HIV DNA in Trex1^−/− MEFs. *, P <0.01, Student's t-test. Error bars indicate S.D. of triplicate replicates of two independent experiments.

Fig. 5. TREX1 siRNA treatment induces IFN-α and IFN-β and inhibits HIV replication in primary human immune cells

(a) Scheme of the experiment for (b-e). Human monocyte-derived macrophages (MDM) were transfected with control, TREX1 or TREX1 plus IRF3 siRNAs and infected 3 days later with HIV_BaL. (b) TREX1 and IRF3 mRNAs were measured 2 days after siRNA transfection by qRT-PCR. (c) Cytosolic HIV DNA increases in MDM treated with TREX1 siRNA, irrespective of co-transfection with IRF3 siRNA. Average values, normalized to values in cells treated with control siRNA, of 4 replicates from 2 donors are shown. (d) TREX1 siRNA inhibits HIV_BaL replication in MDM measured by p24 Gag Ag ELISA of culture supernatants. No measurements were made on day 1 (not determined, nd). (e) HIV infection of MDM transfected with TREX1 siRNA induces IRF3-dependent IFN gene expression. IFN-α and IFN-β mRNA levels were measured by qRT-PCR at indicated days post infection and normalized to the control values on day 1. TREX1 siRNA treatment does not induce IFN in uninfected cells. The late induction of IFN in HIV_BaL-infected cells is likely due to the slow spread of infection in macrophage cultures (only 20% of cells were infected by day 7 by flow cytometry analysis of p24 immunostaining). *, P <0.01, Student's t-test. Error bars indicate S.D. of 4 replicates from 2 independent donors. (f-l) Pretreatment with TREX1 siRNAs induces IFN in HIV_IIIB-infected human CD4⁺ T cells. CD4⁺ T cells, positively selected from PBMC, were transfected with control or TREX1 siRNAs and 3 days later infected with HIV_IIIB. The control (CTL) siRNA was used at 600 pmole and TREX1 siRNA was used at 200, 400 and 600 pmole per 3 × 10⁶ cells. TREX1 mRNA was measured 2 d post transfection to assess siRNA suppression (f). Cytosolic HIV DNA was measured 10 hpi (g); IFN-α (h) and IFNβ (i) mRNA were measured 22 hpi and normalized to the CTL cells. (j) Suppression of TREX1 expression inhibits HIV_IIIB replication in CD4⁺ T cells analyzed 3 days post infection by flow cytometry analysis of p24 immunostaining. A representative analysis is shown in (e) and mean percentage of p24⁺ cells (f), and p24 mean fluorescence intensity (MFI) (g) from six replicates in two independent experiments are shown. *, P <0.05, Student's t-test. Error bars indicate S.D..

Fig. 6. HIV-stimulated IFN induction requires IRF3, TBK1 and STING

(a) Inhibition of selected innate immune genes by siRNA transfection of Trex1^−/− MEFs assessed by qRT-PCR 48 h post transfection. *, P <0.01. Student's t-test. Error bars indicate S.D. of 3 independent experiments. (b) IRF3, TBK1 and STING are required for HIV-stimulated IFN-β induction in Trex1^−/− cells. WT and Trex1^−/− MEFs transfected with indicated siRNAs were infected with VSV-G-pseudotyped HIV and IFN-β mRNA was measured 24 hpi by qRT-PCR. The fold increase compared to si-CTL is displayed. *, P <0.01. Student's t-test. Error bars indicate S.D. of 3 independent experiments. (c) Innate immune factors involved in HIV-stimulated IFN response do not affect HIV DNA accumulation. Cytosolic HIV DNA in Trex1^−/− MEFs transfected with the indicated siRNA was measured 10 hpi by qPCR. See Supplementary Fig. 4 for a model of the innate immune pathway stimulated by HIV DNA.