1. Volume 58, Issue 1, Pages 134-146 (April 2015)
Viral Pseudo-Enzymes Activate RIG-I via Deamidation to Evade Cytokine Production 
Shanping He, Jun Zhao, Shanshan Song, Xiaojing He, Arlet Minassian, Yu Zhou, Junjie Zhang, Kevin Brulois, Yuqi Wang, Jackson Cabo, Ebrahim Zandi, Chengyu Liang, Jae U. Jung, Xuewu Zhang, Pinghui Feng 
Molecular Cell 
Volume 58, Issue 1, Pages (April 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 58, 134-146DOI: (10.1016/j.molcel.2015.01.036)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 RIG-I Is Critical for γHV68 to Evade Cytokine Production
(A) Diagram summarizing the requirement of MAVS and IKKβ to induce RelA degradation and evade cytokine production by γHV68.
(B) MEFs of indicated genotypes were infected with γHV68 (moi = 10). Whole-cell lysates were prepared and analyzed by immunoblotting.
(C and D) Rig-i+/+ and Rig-i−/− MEFs were harvested, and total RNA was extracted. cDNA was analyzed by real-time PCR (C); supernatants were harvested and IL-6 was determined by ELISA (D). ∗∗p < 0.01, ∗∗∗p <
(E) 293T/Flag-RIG-I stable cell line was mock infected or infected with γHV68 (moi = 5) or Sendai virus (SeV, 100 U/ml) for 3 hr (hpi). Purified RIG-I was analyzed by gel filtration and immunoblotting with anti-Flag antibody (left). Whole-cell lysates were analyzed by immunoblotting for exogenous RIG-I and γHV68 TK (ORF21) expression (right).
Error bars denote SD (n = 3). See also Figure S1.
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4. Figure 2 vGAT Interacts with RIG-I
(A) 293T cells were transfected with a reporter cocktail and plasmids containing individual γHV68 genes, and NF-κB activation was determined by luciferase assay. Viral proteins that function during the immediate early phase are shown. IE, proteins expressed in the immediately early phase.
(B) vGAT and its interacting proteins were purified from transfected 293T cells, analyzed by SDS-PAGE, and identified by mass spectrometry.
(C) NIH 3T3/Flag-RIG-I cells were infected with γHV68 (moi = 5) for 16 hr. Whole-cell lysates (WCLs) were precipitated with anti-Flag (RIG-I). Precipitated proteins and WCLs were analyzed by immunoblotting.
(D and E) The structural domains of RIG-I (D) and vGAT (E) are diagrammed (top). 293T cells were transfected with plasmids containing the indicated genes. WCLs were precipitated with anti-Flag (RIG-I and vGAT). Precipitated proteins and WCLs were analyzed by immunoblotting.
(F) GST pull down, with GST or GST-RIG-I-N purified from E.coli and the GAT domain of vGAT translated in vitro, was analyzed by autoradiography (top) and Coomassie staining (bottom). n.s., non-specific.
(G) The interaction between the tandem CARDs of RIG-I and the GAT domain of vGAT.
Error bars denote SD (n = 3). See also Figure S2.
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5. Figure 3 vGAT Directly Activates RIG-I
(A and B) 293T cells stably expressing control or RIG-I shRNA were harvested, and WCLs were analyzed by immunoblotting (A). RIG-I knockdown cells were transfected with NF-κB reporter cocktail and a plasmid containing vGAT or infected with Sendai virus (SeV) (100 HAU for 16 hr). NF-κB activation was determined by luciferase assay (B).
(C) 293T cells were transfected with plasmids containing the indicated genes. GST or GST-RIG-I-N was precipitated in RIPA buffer. Precipitated proteins and WCLs were analyzed by immunoblotting.
(D) 293T cells were transfected with a plasmid containing Flag-tagged ubiquitin. At 24 hr post-transfection, cells were infected with γHV68 wild-type or γHV68ΔvGAT (moi = 10) for 16 hr. WCLs in RIPA buffer were precipitated with anti-RIG-I antibody. Precipitated RIG-I and WCLs were analyzed by immunoblotting with the indicated antibodies.
(E) 293T cells were transfected with plasmids containing the indicated genes. Purified RIG-I was analyzed by gel filtration and immunoblotting (left). Purified RIG-I (5%) and WCLs were analyzed by Coomassie blue staining and immunoblotting, respectively (right).
(F) NIH 3T3/RIG-I stable cells were mock infected or infected with wild-type γHV68 (moi = 10) or γHV68.ΔvGAT for 16 hr. Purified RIG-I was analyzed by gel filtration. Aliquots (30 μl) of fractions and WCLs were analyzed by immunoblotting.
Error bars denote SD (n = 3). See also Figure S3.
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6. Figure 4 vGAT Induces RIG-I Deamidation
(A) 293T cells were transfected with NF-κB reporter plasmid cocktail plus a plasmid containing vGAT or IKKβ and treated with an inhibitor of glutamine amidotransferase, DON (5 μM), at 6 hr post-transfection. NF-κB activation was determined by luciferase assay. ∗∗p < 0.01.
(B) 293/Flag-RIG-I cells were transfected with a plasmid containing ORF75b or vGAT. WCLs were analyzed by 2D gel electrophoresis and immunoblotting.
(C) RIG-I was purified from transfected 293T cells with or without vGAT and analyzed by tandem mass spectrometry for deamidation. Three deamidated residues were identified. Q10 was converted into E (in red) due to deamidation. Please see Figures S4E and S4F for N245 and N445 that were converted into D.
(D) 293T cells were transfected with plasmids containing RIG-I or the RIG-I triple deamidation mutant (RIG-I-TD) without or with a plasmid containing vGAT. WCLs were analyzed by 2D gel electrophoresis and immunoblotting.
(E) 293T/Flag-RIG-I cells were transfected with a plasmid containing γHV68 (m-vGAT) or KSHV (k-vGAT) GAT, and WCLs were analyzed by 2D gel electrophoresis.
(F) 293/Flag-RIG-I cells were infected with γHV68 (moi = 20) or HSV-1 (moi = 2) for 16 hr or SeV (100 HA U/ml), KSHV (moi = 10), or influenza virus (PR8) (moi = 5) for 2 hr. RIG-I deamidation was analyzed similar to that in (E).
Error bars denote SD (n = 3). See also Figure S4.
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7. Figure 5 The Deamidated RIG-I-TD Activates Innate Immune Signaling
(A) 293T cells were transfected with NF-κB reporter cocktail and plasmids containing the indicated genes, and NF-κB activation was determined by luciferase assay.
(B) 293T cells were transfected with plasmids containing wild-type RIG-I or RIG-I-TD. Total RNA was extracted at 24 hr post-transfection and subjected to reverse transcription and real-time PCR analysis.
(C and D) 293T cells were transfected with plasmids containing wild-type RIG-I or RIG-I-TD. RIG-I and RIG-I-TD were precipitated in RIPA buffer and analyzed by immunoblotting (C). Purified RIG-I and RIG-I-TD were analyzed by gel filtration. Elutions and RIG-I-enriched fractions (30 μl) were analyzed by immunoblotting (D).
(E) RIG-I-N and RIG-I-N-Q10E were purified from bacteria and analyzed by gel filtration. x and y axes denote the elution volume and absorbance unit, respectively.
(F) N245 and N445 are located in close proximity to the ATP-binding site in the helicase domain of the RIG-I structure (PDB ID: 3TMI). N245 and N445, sandwiched by E249 and E448, are coded in green and yellow, respectively. ATP is highlighted in purple.
(G and H) RIG-I and RIG-I-TD were purified from transfected 293T cells and analyzed by immunoblotting and ATP hydrolysis, with or without 5′-triphosphate RNA (100 nM) (G). Purified RIG-I and RIG-I-TD (200 nM) were incubated with 32P-labeled 5′-triphosphate RNA (4 nM), without or with a 200-fold excess of cold 5′-triphosphate RNA. RNA-RIG-I complex was analyzed by PAGE and autoradiography (H).
Error bars denote SD (n = 3). See also Figure S5.
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8. Figure 6 vGAT Recruits Cellular PFAS to Deamidate RIG-I
(A and B) MEFs were infected with γHV68 (moi = 5), and WCLs were precipitated with anti-PFAS antibody (A). 293 cells were infected with γHV68 or γHV68.ΔvGAT (moi = 5) for 16 hr. WCLs were precipitated with anti-RIG-I antibody (B). Precipitated proteins and WCLs were analyzed by immunoblotting.
(C) HEK293/Flag-RIG-I cells were infected with γHV68 (moi = 5) or SeV (100 HA U/ml) for 16 hr. WCLs were precipitated with anti-Flag (RIG-I). Precipitated proteins and WCLs were analyzed by immunoblotting.
(D and E) 293T cells were infected with lentivirus expressing control (CTL) or PFAS shRNA. At 72 hr post-infection, cells were harvested and WCLs were analyzed by immunoblotting (D). Transfection of 293T and NF-κB luciferase reporter assay was performed (E). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001; p values were calculated in relation to transfections with the same amount of vGAT plasmid of the control shRNA group.
(F) 293T cells were transfected, and NF-κB activation was determined by luciferase assay.
(G) GST-RIG-I, PFAS, PFAS-ED, and vGAT were purified from 293T cells to homogeneity and analyzed by silver staining (left). In vitro deamidation of RIG-I was analyzed by 2D gel electrophoresis and immunoblotting (right).
(H) In vitro deamidation was carried out as in (G), except RIG-I-N was used. Spectral count of the deamidated peptide containing Q10E was shown. Data represent two independent experiments. ND, not detected. WT and ED, wild-type and the enzyme-dead mutant of PFAS.
Error bars denote SD (n = 3). See also Figure S6.
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9. Figure 7
PFAS Is Critical for RIG-I Deamidation and Activation to Negate Cytokine Production during γHV68 Infection
(A) 293T cells stably expressing control (CTL, scrambled) or PFAS shRNA were transfected with plasmids containing RIG-I and vGAT. Purified RIG-I was analyzed by gel filtration and immunoblotting (left panels). Purified RIG-I (5%) was analyzed by Coomassie staining (top right). WCLs were analyzed by immunoblotting (right panels).
(B–E) Rig-i+/+ MEFs stably expressing control (CTL) or mPFAS shRNA were harvested, and total RNA was extracted and analyzed by real-time PCR for Pfas mRNA levels (B). MEFs were infected with γHV68 (moi = 5). RNA was extracted and cDNA was prepared to determine Ccl-5 and Il-6 mRNA by real-time PCR analysis (C); supernatant was collected to determine IL-6 by ELISA (D). WCLs were prepared and analyzed by immunoblotting (E). Note that we detected two species of RelA in MEFs infected with lentivirus. The larger RelA species was increased in lentivirus-infected MEFs. ∗∗∗p < for (D).
(F) Infection of Rig-i−/− MEFs reconstituted with wild-type RIG-I or RIG-I-3A and immunoblotting were performed as in (E).
(G) Model of the immune evasion strategy employed by γHV68 entailing RIG-I, MAVS, and IKKβ. γHV68 vGAT dimerizes with cellular PFAS to deamidate RIG-I. Triple deamidations (at Q10, N245, and N445) result in RIG-I activation and subsequent activation of MAVS and IKKβ. Activated IKKβ, together with viral RTA, facilitates RelA degradation by proteasome, thereby negating antiviral cytokine gene expression. ∗ denotes deamidation of RIG-I.
Error bars denote SD (n = 3). See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
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