Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses

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.

Figure 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 5μM) or integrase inhibitor (RAL at 10μM) for 30 min before infection with HIV-GFP. 24 h after infection, cell extracts were analyzed by native gel electrophoresis or SDS-PAGE followed by immunoblotting with indicated antibodies (A) and total RNA was isolated for q-RT-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 IFNβ RNA by q-RT-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.

Figure 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 herpes simplex virus (HSV-1) or Sendai virus (SeV), followed by measurement of IRF3 dimerization. (B) L929 cGAS KO 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 h, followed by IRF3 dimerization assay. (C and D) Similar to (B) except that RNA levels of IFNβ (C) and CXCL10 (D) were measured by q-RT-PCR. As a control, some cells were also infected with Sendai virus for 12 h. The error bars indicate standard deviations of triplicate measurements. N.D: non-detectable.

Figure 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 24h. 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 KO clone #18 and the parental cells, both stably expressing an shRNA against TREX1, were infected with HIV-GFP for 22 h. 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 HPLC using a C18 column, and the abundance of cGAMP was quantitated by mass spectrometry using SRM. (D) Comparison of the MS/MS 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) Monocyte-derived macrophages (MDM) or dendritic cells (MDDC) from a human donor were either untreated or treated with Vpx-VLP for 24 h 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 were measured as described in (A). FACS 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.

Figure 4. Murine leukemia virus and simian immunodeficiency virus activate innate immune responses through cGAS

(A and B) L929 cGAS KO clone#18 and the parental L929 cells stably expressing shRNA against TREX1 or luciferase (control) were infected with MLV-GFP (MOI=2) for 20 h, followed by measurement of IFNβ (A) and CXCL10 (B) RNA by q-RT-PCR. (C and D) Similar to (A and B), except that cells were infected with SIV-GFP (MOI=1.5) for 20 h.