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Cyclic GMP-AMP Synthase Is an Innate Immune Sensor of HIV and Other Retroviruses

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Science  23 Aug 2013:
Vol. 341, Issue 6148, pp. 903-906
DOI: 10.1126/science.1240933

HIV Detection Is a (c)GAS

Despite it being one of the most highly studied viruses, there are still many unknowns when it comes to HIV—including how it triggers the innate immune response. Gao et al. (p. 903, published 8 August) now demonstrate that the DNA sensor cyclic GMP-AMP synthase (cGAS) detects HIV infection. Reverse-transcribed HIV DNA triggers cGAS and downstream activation of antiviral immunity. Detection of HIV, as well as the retroviruses simian immunodeficiency virus and murine leukemia virus, was abrogated in mouse and human cells deficient in cGAS—suggesting that cGAS may be a critical activator of innate immunity in response to retroviral infection.

Abstract

Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse-transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.

Although tremendous advances have been made in our understanding of innate immune recognition of many microbial pathogens (13), relatively little is known about innate immune responses against retroviral infections (4). Retroviruses were thought to trigger weak or no innate immune responses, which were typically measured through the production of inflammatory cytokines and type I interferons. However, recent research has shown that retroviruses such as HIV can trigger innate immune responses, which are normally masked by viral or host factors (58). For example, TREX1 is a cytosolic exonuclease that degrades DNA derived from HIV or endogenous retroelements, thereby preventing the accumulation of cytosolic DNA, which would otherwise trigger innate immunity (9, 10). Loss-of-function mutations of TREX1 in humans have been closely linked to Aicardi-Goutières syndrome (AGS), a lupus-like disease characterized by elevated expression of inflammatory cytokines and interferon-stimulated genes (11).

We have recently identified the enzyme cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase (cGAS) as a cytosolic DNA sensor that triggers the production of type I interferons and other cytokines (12, 13). DNA binds and activates cGAS, which catalyzes the synthesis of a cGAMP isomer from adenosine triphosphate (ATP) and guanosine triphosphate (GTP). This cGAMP isomer, termed 2′3′-cGAMP, which contains both 2′-5′ and 3′-5′ phosphodiester linkages, functions as a second messenger that binds and activates the endoplasmic reticulum protein STING (1417). STING then activates the protein kinases IκB kinase (IKK) and TANK-binding kinase 1 (TBK1), which in turn activate the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF3) to induce interferons and other cytokines (18). Knockdown of cGAS inhibits interferon-β (IFN-β) induction by DNA viruses such as herpes simplex virus 1 (HSV-1) and vaccinia virus (13). Because retroviruses generate complementary DNA from the viral RNA by reverse transcription, we hypothesized that cGAS might detect retroviral DNA and trigger innate immune responses.

We used a single-round HIV-1 virus in which its envelope protein was replaced with the glycoprotein of vesicular stomatitis virus (VSV-G), which allows it to infect a large variety of human and mouse cell types (9). This virus also expresses green fluorescent protein (GFP), which can be used to monitor viral infection. Infection of the human monocytic cell line THP1 with HIV-GFP led to dimerization of IRF3 (fig. S1A), a hallmark of its activation. Phosphorylation of STAT1 at Tyr-701 was also detected after HIV infection (fig. S1A), indicating that the interferon signaling pathway was activated in the virus-infected cells (19). HIV infection led to the induction of IFN-β and the chemokine CXCL10 (fig. S1B), concomitant with the generation of the HIV Gag episomal DNA (fig. S1C). The levels of IFN-β production were proportional to the multiplicity of infection by HIV (fig. S1D). Treatment of HIV-GFP virus with DNase I did not impair its ability to induce IFN-β (fig. S1E), whereas treatment of herring testis DNA (HT-DNA) with DNase I inhibited IFN-β induction (fig. S1F), indicating that IFN-β induction by HIV-GFP was not due to any contaminating DNA. Differentiation of THP1 from monocytes to macrophages by treating the cells with phorbol-12-myristate-13-acetate (PMA) inhibited HIV-GFP infection or replication (fig. S1G) and strongly inhibited IFN-β induction (fig. S1H). Thus, unless otherwise indicated, THP1 cells used in our study were not treated with PMA before HIV infection.

To test whether reverse transcription is required for HIV to activate the innate immune response, we treated THP1 cells with the HIV reverse transcriptase inhibitors azidothymidine (AZT) and nevirapine (NVP). Both inhibitors blocked IRF3 activation and IFN-β induction by HIV (Fig. 1, A and B). In contrast, the HIV integrase inhibitor raltegravir (RAL) did not affect the activation of this pathway. AZT and NVP, even at high concentrations, did not inhibit IFN-β induction by HT-DNA (fig. S2, A to C), indicating that the inhibitory effects of AZT and NVP were due to their specific inhibition of HIV reverse transcription. These results suggest that the reverse-transcribed HIV DNA is the trigger of IRF3 activation and IFN-β production.

Fig. 1 The cGAS-STING pathway mediates innate immune responses against HIV.

(A and B) THP1 cells were treated with the HIV reverse transcriptase inhibitors AZT and NVP (each at 5 μM) or the integrase inhibitor RAL (at 10 μM) for 30 min before infection with HIV-GFP. 24 hours after infection, cell extracts were analyzed by native gel electrophoresis or SDS–polyacrylamide gel electrophoresis, followed by immunoblotting with indicated antibodies (A), and total RNA was isolated for quantitative reverse transcription polymerase chain reaction (qRT-PCR) (B). (C and D) THP1 cells stably expressing an shRNA against human cGAS, STING, or luciferase (control) were infected with HIV-GFP for the indicated time, followed by measurement of IFN-β RNA by qRT-PCR (C) and immunoblotting with the indicated antibodies (D). Error bars indicate standard deviations of triplicate measurements. Unless otherwise indicated, data shown in this and all other figures are representative of at least two independent experiments.

Short-hairpin RNA (shRNA)–mediated knockdown of cGAS or STING in THP1 cells strongly inhibited the induction of IFN-β and CXCL10 and the activation of IRF3 by HIV-GFP (Fig. 1, C and D; and fig. S2, D and E). Control experiments showed that shRNA against luciferase did not inhibit the activation of the pathway and that the shRNA vectors knocked down the intended targets specifically. In particular, the cGAS shRNA knocked down cGAS but not STING (Fig. 1D), and the induction of IFN-β in these cells was rescued by delivering cGAMP into the cells (fig. S2F), indicating that the cGAS shRNA did not have off-target effects in the STING pathway.

Previous studies have shown that VSV-G–pseudotyped HIV-1 strongly induces IFN-β in TREX1-deficient mouse embryonic fibroblasts (MEFs) but not in the wild-type (WT) MEF (9). We generated Trex1−/− MEF cell lines stably expressing shRNA against cGAS, STING, or luciferase (as a control; fig. S3, A and B). HIV infection induced IFN-β and CXCL10 RNA in the control cells (sh-luciferase) but not in cGAS- or STING-depleted cells (fig. S3, C and D). In contrast, knockdown of cGAS or STING did not affect the induction of IFN-β or CXCL10 by the double-stranded RNA analog poly[I:C].

To obtain definitive evidence for the role of cGAS in the innate sensing of cytosolic DNA and retroviruses, we employed the TALEN technology to disrupt the gene that encodes cGAS (Mb21d1); specifically, the region that encodes the catalytic domain, in L929 cells (see methods and fig. S4A) (20). Although L929 cells contain three copies of chromosome 9, which harbors the cGAS gene, DNA sequencing of the TALEN-expressing cells identified multiple clones that had deletions in all three chromosomes; three of these clones were chosen for further studies (fig. S4B). All three clones contained deletions in the cGAS locus that generated frameshift mutations (21).

All three cGAS mutant cell lines failed to activate IRF3 in response to HT-DNA transfection or HSV-1 (a double-stranded DNA virus) infection (Fig. 2A and fig. S4C). As controls, these cells activated IRF3 normally in response to transfection with poly[I:C] or infection with Sendai virus, an RNA virus. The cGAS mutant cells were also defective in inducing CXCL10 in response to HT-DNA, but this defect was rescued by transfecting the cells with the mouse cGAS expression plasmid (fig. S4D).

Fig. 2 cGAS is essential for innate immune responses triggered by HIV.

(A) L929 cell lines harboring various deletions in exon 2 of the cGAS locus were generated by TALEN (see fig. S4). These cells and the parental L929 cells were infected with HSV-1 or Sendai virus (SeV), followed by measurement of IRF3 dimerization. (B) L929 cGAS knockout clone 18 and parental L929 cells were stably transfected with shRNA vector targeting TREX1 or luciferase (as a control). These cells were infected with HIV-GFP for 20 hours, followed by an IRF3 dimerization assay. (C and D) Similar to (B) except that RNA levels of IFN-β (C) and CXCL10 (D) were measured by qRT-PCR. As a control, some cells were also infected with Sendai virus for 12 hours. The error bars indicate standard deviations of triplicate measurements. N.D., nondetectable.

We chose cGAS mutant clone 18 and the parental L929 cells to investigate the role of cGAS in innate immune recognition of HIV infection. In L929 cells stably expressing an shRNA against TREX1, but not the control luciferase, HIV-GFP infection induced IRF3 dimerization and the production of IFN-β and CXCL10 (Fig. 2, B to D; and fig. S4, E and F). In contrast, the L929 cGAS mutant cells failed to mount any detectable immune response to HIV infection even when TREX1 was depleted, demonstrating the essential role of cGAS in immune responses against HIV. The depletion of cGAS did not affect IFN-β or CXCL10 induction by Sendai virus (Fig. 2, C and D).

We have previously shown that HEK293T cells do not express detectable levels of cGAS and STING and thus fail to activate IRF3 in response to DNA transfection or DNA virus infection (13). Consistent with an important role of cGAS and STING in retrovirus detection, HIV-GFP infection activated IRF3 and STAT1 in THP1 but not HEK293T cells (fig. S5A). In contrast, Sendai virus activated IRF3 and STAT1 in both cell lines. To determine whether HIV infection leads to the production of endogenous cGAMP in human cells, we prepared lysates from HIV-infected THP1 and HEK293T cells and heated the lysates at 95°C to denature most proteins, which were removed by centrifugation (12). The supernatant that potentially contained cGAMP was delivered to THP1 cells that had been permeabilized with the bacterial toxin perfringolysin-O (PFO), and then IRF3 dimerization was assayed by native gel electrophoresis (fig. S5B). The heat-resistant supernatant from HIV-infected THP1, but not HEK293T, cells contained the cGAMP activity that stimulated IRF3 activation in the recipient cells. Furthermore, inhibition of HIV reverse transcription by AZT, DDI (didanosine), or NVP blocked the generation of the cGAMP activity, whereas the HIV integrase inhibitor RAL had no effect (Fig. 3A). HIV-GFP infection in L929-shTrex1 cells also led to generation of the cGAMP activity, which was dependent on cGAS (Fig. 3B). Taken together, these results indicate that HIV infection induces the production of endogenous cGAMP in a manner that depends on cGAS and the reverse transcription of HIV RNA to cDNA.

Fig. 3 HIV infection induces the production of cyclic GMP-AMP in human cells.

(A) THP1 cells were treated with inhibitors of HIV reverse transcriptase (AZT, DDI, and NVP) or integrase (RAL) before infection with HIV-GFP for 24 hours. Cell extracts were prepared for native gel electrophoresis to detect endogenous IRF3 dimer (top panel). Aliquots of the cell extracts were treated at 95°C for 5 min to denature proteins, which were subsequently removed by centrifugation. The cGAMP activity in the supernatant was measured after its delivery into PFO-permeabilized THP1 cells, followed by IRF3 dimerization assay (bottom panel). (B) L929 cGAS knockout clone 18 and the parental cells, both stably expressing an shRNA against TREX1, were infected with HIV-GFP for 22 hours. Endogenous IRF3 activation and cGAMP production in the cells were measured as described in (A). (C) The heat-resistant supernatants from THP-1 cells without (mock) or with HIV-GFP infection were fractionated by high-performance liquid chromatography using a C18 column, and the abundance of cGAMP was quantitated by mass spectrometry using SRM. (D) Comparison of the tandem mass spectrometry spectra of cGAMP isolated from HIV-infected THP-1 cells and that synthesized in vitro by recombinant human cGAS protein. Higher-energy collision dissociation (HCD) was used to fragment the precursor ion ([M+H]+ = 675.107,) and normalized collision energy was set at 25. (E) MDMs or MDDCs from a human donor were either untreated or treated with Vpx-VLP for 24 hours before infection with HIV. MDMs were infected with HIV-BaL for 3 days, whereas MDDCs were infected with HIV-GFP for 1 day. The cGAMP activity in these cells was measured as described in (A). Fluorescence-activated cell sorting analysis of HIV infection and measurement of cGAMP abundance by mass spectrometry are shown in fig. S6. The data are representative of three independent experiments involving three human donors.

To test whether HIV infection produces retroviral cDNA in the cytoplasm to activate cGAS, we infected HEK293T cells with HIV-GFP and prepared cytosolic extracts that were then incubated with purified cGAS protein in the presence of ATP and GTP (fig. S5C). Cytosolic extracts from HIV-infected cells, but not from uninfected cells, were able to stimulate cGAS to produce the cGAMP activity that activated IRF3 in permeabilized THP1 cells. Treatment of HEK293T cells with AZT inhibited the generation of the cGAS stimulatory activity. Further analyses showed that the cytoplasm of HIV-infected cells contained the HIV Gag DNA (fig. S5D) and GFP protein (fig. S5E), both of which were inhibited by AZT.

Quantitative measurement of cGAMP abundance by mass spectrometry using selective reaction monitoring (SRM) provided the direct evidence that cGAMP was produced in HIV-infected, but not mock-treated, THP1 cells (Fig. 3C). Tandem mass spectrometry of the endogenous cGAMP from HIV-infected THP1 cells revealed that it was identical to the cGAS product, 2′3′-cGAMP (Fig. 3D) (15).

To test whether HIV infection in primary human immune cells leads to cGAMP production, we infected monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) with the clinical HIV-1 isolate HIV-BaL and HIV-GFP, respectively. Previous research has shown that human macrophages and dendritic cells express SAMHD1, a nuclease that hydrolyzes dNTP, thereby inhibiting HIV reverse transcription (7, 8). HIV-2 and simian immunodeficiency virus (SIV) contain the protein Vpx, which targets SAMHD1 for ubiquitin-mediated proteasomal degradation, thus removing this host restriction factor. To facilitate HIV infections in human MDMs and MDDCs, we delivered the SIV Vpx into these cells using a virus-like particle (VLP) before HIV infection (fig. S6, A, B, and D) (6). In the presence of Vpx, infection of MDMs and MDDCs with HIV-BaL and HIV-GFP, respectively, led to the generation of cGAMP activity (Fig. 3E). Quantitative mass spectrometry analysis further confirmed the production of 2′3′-cGAMP in HIV-infected MDDCs that expressed Vpx (fig. S6C). The cGAMP activity was consistently observed in MDDCs and MDMs of additional human donors, and this activity was higher in the cells infected with HIV than in those treated with Vpx alone (fig. S6, D to F). These results demonstrate that HIV infection in human macrophages and dendritic cells leads to the generation of cGAMP under conditions that are permissive to viral replication.

Finally, we tested whether cGAS is required for innate immune responses against other retroviruses by infecting L929 and L929-cGAS knockout cell lines with murine leukemia virus (MLV) and SIV. Similar to HIV, MLV and SIV induced IFN-β and CXCL10 RNA in L929 cells depleted of endogenous TREX1, but such induction was completely abolished in the cGAS knockout cells (Fig. 4A-4D). In further support of an essential role of the cGAS-STING pathway in innate immune sensing of retroviruses, knockdown of cGAS or STING in Trex1−/− MEF cells strongly inhibited IFN-β induction by MLV and SIV (fig. S7, A to D).

Fig. 4 MLV and SIV activate innate immune responses through cGAS.

(A and B) L929 cGAS knockout (KO) clone 18 and the parental L929 cells stably expressing shRNA against TREX1 or luciferase (control) were infected with MLV-GFP [multiplicity of infection (MOI) = 2] for 20 hours, followed by measurement of IFN-β (A) and CXCL10 (B) RNA by qRT-PCR. (C and D) Similar to (A) and (B), except that cells were infected with SIV-GFP (MOI = 1.5) for 20 hours.

Here we have demonstrated that cGAS is essential for innate immune responses against HIV, SIV, and MLV, suggesting that cGAS is a general innate immune sensor of retroviral DNA. Although HIV primarily infects human CD4 T cells, it can also enter macrophages and dendritic cells, normally without triggering an overt innate immune response by concealing the viral nucleic acids within the capsid and by limiting the accumulation of viral DNA through co-opting host factors such as TREX1 and SAMHD1 (8). The absence of a rigorous innate immune response to HIV in dendritic cells is thought to be a major factor that hampers productive T cell responses and vaccine development (7). Our finding that HIV and other retroviruses can induce the production of cGAMP through cGAS under permissive conditions suggests that cGAMP might be used to bypass the block of innate immune responses against HIV. As such, cGAMP could be a candidate vaccine adjuvant for HIV and other pathogens that are adept at subverting the host innate immune system.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1240933/DC1

Materials and Methods

Figs. S1 to S7

References

References and Notes

  1. Clone 18 has frameshift mutations in all three chromosomes. In addition to frameshifts, clone 36 harbored a 9–base pair (bp) deletion in one chromosome that removed three amino acids (215MFK217) in the catalytic domain, whereas clone 94 had a 12-bp deletion in one chromosome and an 18-bp deletion in another that removed four (214VMFK217) and six (212FDVMFK217) amino acids in the catalytic domain, respectively. For details, see materials and methods in the supplementary materials.
  2. Acknowledgments: We thank X. Chen for assistance with mass spectrometry. The data presented in this manuscript are tabulated in the main paper and in the supplementary materials. Materials including the cGAS knockout cell lines may be requested upon signing of a materials transfer agreement. This work was supported by NIH grants to Z.J.C. (R01-AI093967) and N.Y. (RO1-AI098569). Z.J.C is an investigator of the Howard Hughes Medical Institute.
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