Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

Abstract

Infection of cells with DNA viruses triggers innate immune responses mediated by DNA sensors. cGMP-AMP synthase (cGAS) is a key DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which binds to and activates stimulator of interferon genes (STING), leading to IFN production and an antiviral response. Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA virus that is linked to several human malignancies. We report that KSHV infection activates the cGAS-STING pathway, and that cGAS and STING also play an important role in regulating KSHV reactivation from latency. We screened KSHV proteins for their ability to inhibit this pathway and identified six viral proteins that block IFN-β activation through this pathway. This study is the first report identifying multiple viral proteins encoded by a human DNA virus that inhibit the cGAS-STING DNA sensing pathway. One such protein, viral interferon regulatory factor 1 (vIRF1), targets STING by preventing it from interacting with TANK binding kinase 1 (TBK1), thereby inhibiting STING's phosphorylation and concomitant activation, resulting in an inhibition of the DNA sensing pathway. Our data provide a unique mechanism for the negative regulation of STING-mediated DNA sensing. Moreover, the depletion of vIRF1 in the context of KSHV infection prevented efficient viral reactivation and replication, and increased the host IFN response to KSHV. The vIRF1-expressing cells also inhibited IFN-β production following infection with DNA pathogens. Collectively, our results demonstrate that gammaherpesviruses encode inhibitors that block cGAS-STING-mediated antiviral immunity, and that modulation of this pathway is important for viral transmission and the lifelong persistence of herpesviruses in the human population.

Keywords: KSHV; STING; cGAS; innate immunity; vIRF1.

Fig. 1.

KSHV primary infection can activate a STING-cGAS–dependent IFN-β response. IFN-β mRNA in HUVECs (A) or EA.hy926 endothelial cells (B) was measured by real-time qPCR 4 h after transfection of various DNA fragments (ISD90, HSV60, and E. coli DNA at 5 μg/mL) and cGAMP (5 μg/mL) with Lipofectamine 2000 (Life Technologies). The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the treated and mock samples was calculated. IFN-β in HUVECs (C) or EA.hy926 endothelial cells (D) was measured by ELISA 24 h after the same treatment described in A and B. (E) HUVECs or EA.hy926 cells were infected by HSV-1 at an MOI of 10, and IFN-β mRNA levels were measured 4 h postinfection (hpi) by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the uninfected and HSV-1–infected samples was calculated. (F) HUVECs or EA.hy926 cells were infected with HSV-1 at an MOI of 10, and IFN-β protein levels were measured 24 hpi by ELISA. HUVECs were treated with NS, STING (G), or cGAS (H) siRNA for 72 h. The treated cells were then infected with KSHV (30 genome copies per cell) for 8 h before IFN-β mRNA was measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the siSTING or sicGAS sample compared with the siNS sample was calculated. Knockdown efficiency of STING and cGAS was monitored by real-time qPCR, and their mRNA levels were normalized to the actin mRNA level in each sample. EA.hy926 cells were treated with NS, STING (I), or cGAS (J) siRNA for 72 h. The treated cells were then infected with KSHV (30 genome copies per cell) for 8 h before IFN-β mRNA levels were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the siSTING or sicGAS sample compared with the siNS sample was calculated. Knockdown efficiency of STING or cGAS was monitored by real-time qPCR, and their mRNA levels were normalized to the actin mRNA level in each sample. HUVECs (K) and EA.hy926 cells (L) were treated with NS, STING, or cGAS siRNA for 72 h. Cell lysates were harvested, and endogenous STING and cGAS were detected by Western blot analysis. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01 (both by Student’s t test).

Fig. S1.

Induction of IFN-β by KSHV primary infection. (A) HUVECs were treated with NS, STING, or cGAS siRNAs for 72 h and then infected with KSHV for 24 h before performing an IFN-β ELISA. EA.hy926 cells were transduced with lentivirus-based shRNA targeting NS, STING (B), or cGAS (C). Cells were then infected with KSHV (30 DNA genome copies per cell) for 8 h before IFN-β mRNA was measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the siSTING or sicGAS sample compared with the siNS sample was calculated. Knockdown efficiency of STING and cGAS was monitored by real-time qPCR, and their mRNA levels were normalized to the actin mRNA level in each sample. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01.

Fig. 2.

KSHV DNA motif induces a STING-cGAS–dependent IFN-β response. HUVECs (A) or EA.hy926 cells (B) were transfected with DR1, DR2, or KSHV120 fragments by Lipofectamine 2000 for 4 h before IFN-β mRNA levels were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference of the transfected samples compared with the mock sample was calculated. HUVECs (C) or EA.hy926 cells (D) treated with NS, STING, or cGAS siRNA were transfected with KSHV120 for 4 h before cells were lysed for immunoblot analysis. HUVECs were treated with NS, STING (E), or cGAS (F) siRNA for 72 h and then transfected with KSHV120 for 4 h before IFN-β mRNA levels were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the siSTING or sicGAS sample compared with the siNS sample was calculated. STING or cGAS knockdown efficiency was monitored by real-time qPCR, and their mRNA levels were normalized to the actin mRNA level in each sample. EA.hy926 cells were treated with NS, STING (G), or cGAS (H) siRNA for 72 h and then transfected with KSHV120 for 4 h before IFN-β mRNA levels were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the siSTING or sicGAS sample compared with the siNS sample was calculated. STING or cGAS knockdown efficiency was monitored by real-time qPCR, and their mRNA levels were normalized to the actin mRNA level in each sample. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; ** P < 0.01 (both by Student’s t test).

Fig. S2.

Induction of IFN-β by a KSHV DNA motif. (A) Sequences of DR1, DR2, and KSHV120. (B) EA.hy926 cells treated with NS, STING, or cGAS siRNA were transfected with KSHV120 for 24 h before supernatants were collected for IFN-β ELISA. (C) HEK293T cells overexpressing HA-STING or FLAG-cGAS were lysed and pulled down with either streptavidin beads only or beads with biotin-KSHV120. Immunoblots were performed using HA or FLAG antibody accordingly. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01.

Fig. 3.

KSHV reactivation activates a cGAS-STING–dependent IFN-β response. (A) iSLK.219 cells were transfected with NS, STING, or cGAS siRNA for 72 h and then treated with Dox for various time periods. GFP and RFP were monitored at 48 h and 72 h post-Dox treatment. (B and C) IFN-β mRNA levels 72 h post-Dox treatment from A were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the siSTING or sicGAS sample compared with the siNS sample was calculated. Knockdown efficiency of STING (B) or cGAS (C) was monitored by real-time qPCR, and their mRNA levels were normalized to the actin mRNA level in each sample. (D) IFN-β ELISA was performed 72 h postreactivation with Dox. (E) Immunoblot analysis of cell lysate 48 h post-Dox treatment of A. (F) RNA was extracted from iSLK.219 cells 72 h post-Dox treatment of A, transcription of KSHV viral genes was monitored using real-time qPCR, and their mRNA levels were normalized to the actin mRNA level in each sample. (G) iSLK.219 cells were treated as described in the main text. At 72 h post-Dox treatment, RNA was extracted from duplicate samples and KSHV viral transcript levels were analyzed using a KSHV real-time qPCR-based whole-genome array. mRNA levels of viral genes were normalized to the mRNA levels of multiple cellular housekeeping genes to yield delta cycle threshold (dCT) as a measure or relative expression. These values were then subjected to unsupervised clustering. A heat map and dendrogram depicted by the brackets is shown. As shown in the key, higher transcript levels are indicated by red and lower levels are indicated by blue. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01 (both by Student’s t test). (Also Fig. S3B.)

Fig. S3.

Depletion of cGAS and STING augments KSHV reactivation and gene expression. (A) iSLK219 cells were transfected with NS, STING, or cGAS siRNA for 72 h and then treated with DOX for various time points. RFP-positive cells were quantitated using ImageJ (NIH). (B) iSLK219 cells were treated as described in the main text. At 72 h post-DOX treatment, total RNA was extracted and KSHV viral transcript levels were analyzed using a KSHV viral array. mRNA levels of viral genes were normalized to the mRNA levels of multiple cellular housekeeping genes. A heat map for the viral array is shown. Higher transcript levels are indicated by red, and lower levels are indicated by blue.

Fig. 4.

Screening of KSHV ORFs that modulate the cGAS-STING–dependent pathway. (A) HEK293T cells were cotransfected with 50 ng of IFN-β promoter luciferase and various plasmids (pCDNA3, 2.5 ng of pCDNA-STING-HA, or 50 ng of pUNO-cGAS or pCDNA-STING-HA and pUNO-cGAS combined). Luciferase activity was measured 36 h posttransfection in the cell lysates. A CMV-driven Renilla plasmid was cotransfected as a transfection control. (B) Schematic of cGAS-STING–based screening. Cells were transfected with the same amount of STING and cGAS expression plasmid, plus 100 ng of KSHV ORF expression plasmid or EV. (C) Waterfall plot of the effect of KSHV ORFs on cGAS-STING– based screening. The top six inhibitors and one activator are shown. (D) Heat map of the effect of KSHV ORFs on the cGAS-STING pathway. Higher IFN-β promoter luciferase activation levels are indicated by red, whereas lower levels are indicated by blue, which corresponds to a higher degree of inhibition. The six inhibitors are marked with a bracket. (E) Top six KSHV ORF inhibitor expression plasmids were cotransfected with STING and cGAS expression plasmids. Thirty-six hours later, IFN-β mRNA levels were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold changes in IFN-β mRNA levels compared with the vector control are displayed on the y axis. (F) Top six KSHV ORF inhibitor expression plasmids were cotransfected with STING and cGAS expression plasmids, and IFN-β protein levels were measured by ELISA 36 h posttransfection. (G) K13 expression plasmid was cotransfected with STING and cGAS expression plasmids, and IFN-β mRNA levels were measured by real-time qPCR 36 h posttransfection. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold change between the K13-expressing vs. vector-expressing cells was calculated. (H) K13 was cotransfected with STING and cGAS plasmids, and IFN-β protein levels were measured by ELISA 36 h posttransfection. (I) Varying doses of the top six KSHV ORF inhibitor expression plasmids (25 ng, 50 ng, or 100 ng) were cotransfected with STING and cGAS expression plasmids, and IFN-β promoter luciferase activity was measured 36 h posttransfection. (J) Varying doses of a K13 expression plasmid (25 ng, 50 ng, or 100 ng) were cotransfected with STING and cGAS expression plasmids, and IFN-β promoter-driven luciferase activity was measured 36 h posttransfection. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01 (both by Student’s t test).

Fig. 5.

KSHV vIRF1 inhibits cGAS-STING sensing and promotes HSV-1 replication. HUVECs or EA.hy926 endothelial cells were transduced with EV or vIRF1-expressing lentivirus to generate EV or vIRF1-expressing cells. These cells were used in the following experiments unless otherwise noted. (A) IFN-β mRNA levels in transduced HUVECs were measured by real-time qPCR 4 h posttransfection of various DNA fragments (ISD90, HSV60, VACV70, KSHV120, and E. coli DNA at 5 μg/mL). The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold differences between the treated samples compared with the mock samples were calculated. (B) IFN-β protein levels in HUVEC transduced cells were measured by ELISA 24 h posttransfection of various DNA fragments (ISD90, HSV60, VACV70, KSHV120, and E. coli DNA at 5 μg/mL). (C) Transduced EA.hy926 cells were treated, and IFN-β mRNA levels were measured as in A. (D) Transduced EA.hy926 cells were treated, and IFN-β protein levels were measured as in B. (E) Transduced HUVECs were infected by HSV-1 at an MOI of 10. IFN-β mRNA levels in these cells were measured by real-time qPCR 4 hpi. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the HSV-1–infected samples compared with the uninfected mock samples was calculated. (F) Transduced HUVECs were infected by HSV-1 at an MOI of 10. IFN-β protein levels were also measured by ELISA 24 hpi. (G) Transduced EA.hy926 cells were treated and IFN-β mRNA was measured as in E. (H) Transduced EA.hy926 cells were treated and IFN-β protein levels were measured as in F. Transduced HUVECs (I) or EA.hy926 cells (J) were infected with HSV-1 at various MOIs (0.01, 0.1, or 1). At 24 or 48 hpi, supernatants were subjected to a plaque assay to obtain the HSV-1 viral titer. (K) Cells from J were monitored by bright-field microscopy 24 hpi. (L) vIRF1 protein levels were monitored by immunoblotting in HUVECs or EA.hy926 cells transduced with EV or vIRF1-expressing lentivirus. Data are presented as mean ± SD from at least three independent experiments. * P < 0.05; **P < 0.01 (both by Student’s t test).

Fig. 6.

Loss of KSHV vIRF1 results in elevated IFN-β production and attenuated KSHV reactivation and replication. The iSLK.219 cells were transfected with either vIRF1 or NS siRNA for 24 h and then treated with Dox for 24 h. (A) IFN-β mRNA levels were measured by qPCR. (B) IFN-β protein levels were measured by ELISA. (C) vIRF1 mRNA levels were measured by qPCR. (D) Transcription of KSHV viral genes was monitored using real-time qPCR. (E) GFP and RFP were monitored at 48 h and 72 h post-Dox treatment. (F) Average RFP intensities were calculated, and the reading of the siNS group at 48 h was set as 100%. Other groups were normalized to siNS group. (G) Immunoblot analysis of vIRF1 using cell lysate 48 h and 72 h post-Dox treatment. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01 (both by Student’s t test).

Fig. 7.

KSHV vIRF1 inhibits the cGAS-STING DNA sensing pathway. HUVECs or EA.hy926 endothelial cells were transduced with EV or vIRF1-expressing lentivirus to generate EV- or vIRF1-expressing cells. These cells were used in the following experiments unless otherwise noted. (A) Transduced HUVECs were transfected with cGAMP (5 μg/mL) with Lipofectamine 2000 for 4 h before cells were harvested, and IFN-β mRNA levels were measured by real-time qPCR. The relative amount of IFN-β mRNA was normalized to the 18S ribosomal RNA level in each sample, and the fold difference between the cGAMP-transfected samples compared with the mock samples was calculated. (B) Transduced HUVECs were transfected with cGAMP (5 μg/mL) with Lipofectamine 2000 for 24 h before supernatants were harvested, and IFN-β protein levels were measured by ELISA. (C) Transduced EA.hy926 cells were treated, and IFN-β mRNA levels were measured as in A. (D) Transduced EA.hy926 cells were treated, and IFN-β protein levels were measured as in B. Transduced HUVECs (E) or EA.hy926 cells (F) were transfected with ISD90 for 0, 3, 6, and 9 h before harvest. Cells were lysed for immunoblot analysis. (G and H) Coimmunoprecipitation of HA-STING and FLAG-vIRF1 in HEK293T cells. HA-STING and FLAG-vIRF1 expression plasmids were cotransfected into HEK293T cells, followed by coimmunoprecipitation for STING using HA antibody or for vIRF1 using FLAG antibody. STING coimmunoprecipitates with vIRF1 (G), and vIRF1 coimmunoprecipitates with STING (H). (I) Coimmunoprecipitation of myc-vIRF1 and endogenous STING in EA.hy926-vIRF1 stable cells. Cell lysates were precipitated with anti-myc antibody and subjected to immunoblotting. (J) Coimmunoprecipitation of HA-STING and FLAG-TBK1 in HEK293T cells cotransfected with HA-STING and FLAG-TBK1 expression plasmids, with different doses of vIRF1 expression plasmid. Twenty-four hpi, protein lysates were subjected to coimmunoprecipitation and the immunoprecipitates were immunoblotted for the presence of STING, vIRF1, and TBK1 as indicated. (K) Coimmunoprecipitation of STING-V5 and endogenous TBK1 in 293T-STING-V5 stable cells in the presence or absence of myc-vIRF1. Cell lysates were precipitated with V5 antibody and subjected to immunoblotting. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05; **P < 0.01 (both by Student’s t test).

Fig. S4.

Interaction of vIRF1 and STING. (A) 293T cells were transfected with the indicated plasmids and an IFN-β promoter luciferase reporter plasmid, and luciferase assays were performed 36 h after transfection. (B) 293T cells were transfected with the indicated plasmids and an IFN-β promoter luciferase reporter plasmid, and luciferase assays were performed 36 h after transfection. Forty-eight–well plates were used. Each well contained the following amounts of plasmid as labeled: IFN-β luciferase (IFNβ-luc) = 50 ng, pRL-CMV = 5 ng, STING = 2.5 ng, cGAS = 100 ng, TBK1/IRF3sa = 50 ng, and TBK1*2/IRF3sa*2 = 100 ng. (C) Coimmunoprecipitation of STING-HA mutants and myc-vIRF1 in HEK293T cells. Plasmids were cotransfected into HEK293T cells, followed by coimmunoprecipitation for STING mutants using an anti-HA antibody or for vIRF1 using an anti-myc antibody. (D) Coimmunoprecipitation (IP) of STING-HA and FLAG-vIRF1 mutants in HEK293T cells. Plasmids were cotransfected into HEK293T cells, followed by coimmunoprecipitation for STING mutants using anti-HA antibody or for vIRF1 using anti-FLAG antibody.

Fig. S5.

vIRF1 does not block STING trafficking. vIRF1-myc and pUNO-cGAS were transfected as indicated in 293T-STING-V5 stable cells. Twenty-four hours later, cells were fixed and stained.