Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

doi: 10.1073/pnas.1503831112

Figure Lengend Snippet: 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).

Article Snippet: IFN-β promoter luciferase was a generous gift from Zhijian Chen, University of Texas Southwestern, Dallas. pUNO-IRF3sa was obtained from Invivogen. pcDNA3 vIRF1-FLAG was generated in our laboratory as described previously ( 21 ). pcDNA3 FLAG-vIRF1-N (1–224 aa) and pcDNA3 FLAG-vIRF1-C (200–449 aa) were generated in our laboratory based on a codon-optimized vIRF1 (created by Genescript and cloned into pcDNA3.1).

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Transfection, Expressing, Activation Assay, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

doi: 10.1073/pnas.1503831112

Figure Lengend Snippet: 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).

Article Snippet: IFN-β promoter luciferase was a generous gift from Zhijian Chen, University of Texas Southwestern, Dallas. pUNO-IRF3sa was obtained from Invivogen. pcDNA3 vIRF1-FLAG was generated in our laboratory as described previously ( 21 ). pcDNA3 FLAG-vIRF1-N (1–224 aa) and pcDNA3 FLAG-vIRF1-C (200–449 aa) were generated in our laboratory based on a codon-optimized vIRF1 (created by Genescript and cloned into pcDNA3.1).

Techniques: Transduction, Expressing, Enzyme-linked Immunosorbent Assay, Infection, Plaque Assay, Microscopy, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

doi: 10.1073/pnas.1503831112

Figure Lengend Snippet: 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).

Article Snippet: IFN-β promoter luciferase was a generous gift from Zhijian Chen, University of Texas Southwestern, Dallas. pUNO-IRF3sa was obtained from Invivogen. pcDNA3 vIRF1-FLAG was generated in our laboratory as described previously ( 21 ). pcDNA3 FLAG-vIRF1-N (1–224 aa) and pcDNA3 FLAG-vIRF1-C (200–449 aa) were generated in our laboratory based on a codon-optimized vIRF1 (created by Genescript and cloned into pcDNA3.1).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

doi: 10.1073/pnas.1503831112

Figure Lengend Snippet: 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).

Article Snippet: IFN-β promoter luciferase was a generous gift from Zhijian Chen, University of Texas Southwestern, Dallas. pUNO-IRF3sa was obtained from Invivogen. pcDNA3 vIRF1-FLAG was generated in our laboratory as described previously ( 21 ). pcDNA3 FLAG-vIRF1-N (1–224 aa) and pcDNA3 FLAG-vIRF1-C (200–449 aa) were generated in our laboratory based on a codon-optimized vIRF1 (created by Genescript and cloned into pcDNA3.1).

Techniques: Transduction, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Western Blot, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

doi: 10.1073/pnas.1503831112

Figure Lengend Snippet: 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.

Article Snippet: IFN-β promoter luciferase was a generous gift from Zhijian Chen, University of Texas Southwestern, Dallas. pUNO-IRF3sa was obtained from Invivogen. pcDNA3 vIRF1-FLAG was generated in our laboratory as described previously ( 21 ). pcDNA3 FLAG-vIRF1-N (1–224 aa) and pcDNA3 FLAG-vIRF1-C (200–449 aa) were generated in our laboratory based on a codon-optimized vIRF1 (created by Genescript and cloned into pcDNA3.1).

Techniques: Transfection, Luciferase, Plasmid Preparation, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses

doi: 10.1073/pnas.1503831112

Figure Lengend Snippet: 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.

Article Snippet: IFN-β promoter luciferase was a generous gift from Zhijian Chen, University of Texas Southwestern, Dallas. pUNO-IRF3sa was obtained from Invivogen. pcDNA3 vIRF1-FLAG was generated in our laboratory as described previously ( 21 ). pcDNA3 FLAG-vIRF1-N (1–224 aa) and pcDNA3 FLAG-vIRF1-C (200–449 aa) were generated in our laboratory based on a codon-optimized vIRF1 (created by Genescript and cloned into pcDNA3.1).

Techniques: Blocking Assay, Transfection, Staining