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. Author manuscript; available in PMC: 2016 Apr 2.
Published in final edited form as: Mol Cell. 2015 Mar 5;58(1):134–146. doi: 10.1016/j.molcel.2015.01.036

Viral Pseudo Enzymes Activate RIG-I via Deamidation to Evade Cytokine Production

Shanping He 1, Jun Zhao 1, Shanshan Song 1, Xiaojing He 2, Arlet Minassian 1, Yu Zhou 1, Junjie Zhang 1, Kevin Brulois 1, Yuqi Wang 1, Jackson Cabo 1, Ebrahim Zandi 1, Chengyu Liang 1, Jae U Jung 1, Xuewu Zhang 2, Pinghui Feng 1,*
PMCID: PMC4385502  NIHMSID: NIHMS670019  PMID: 25752576

SUMMARY

RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologues of phosphoribosylformyglycinamide synthase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homologue thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme.

INTRODUCTION

Innate immunity is the first line of defense against invading pathogens. Upon infection, pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs) within distinct cellular compartments (Takeuchi and Akira, 2010). The retinoic acid-induced gene I (RIG-I) and melanoma differentiation antigen 5 (MDA5) proteins are cytosolic sensors that detect viral RNA through the carboxyl terminal domain (CTD) (Hornung et al., 2006; Kato et al., 2006; Pichlmair et al., 2006; Schmidt et al., 2009). In resting cells, RIG-I and MDA5 are kept in an inert state via an intramolecular interaction between the N-terminal CARDs and the internal helicase domain (Saito et al., 2007; Takahasi et al., 2008). Recent structural studies revealed that RNA-binding perturbs this auto-inhibitory interaction, releasing the N-terminal tandem CARDs (Kowalinski et al., 2011; Luo et al., 2011). The exposed CARDs trigger oligomerization of RIG-I and its MAVS adaptor (also known as IPS-1, VISA, and CARDIF), enabling downstream gene expression via activating NF-κB and interferon regulatory factors (IRF) (Fitzgerald et al., 2003; Sharma et al., 2003). These signaling cascades constitute potent innate immune responses that establish an anti-viral state during the early stages of infection. In essence, activation of RIG-I and MDA5 by viral RNA is central to innate immune defense and represent a paradigm concerning the activation of pattern recognition receptors. It is not clear whether pathogen component other than RNA can activate RIG-I or receptors alike.

Emerging studies indicate that bacterial effectors possess intrinsic activity to deamidate key signaling molecules and manipulate host innate immune defenses (Cui et al., 2010). Glutamine amidotransferase (GAT) is a deamidase participating in the biosynthesis of a wide array of metabolites, including amino acids, nucleotides, lipids and enzyme cofactors (Zrenner et al., 2006). Phosphoribosyl-formylglycinamide synthase (PFAS) catalyzes the fourth step of the purine synthesis pathway. Although protein deamidation was reported half a century ago (Mycek and Waelsch, 1960), it is largely regarded as a spontaneous, nonspecific process of protein degradation. Recent studies found that functions of Bcl-XL and 4EBP2 were controlled via deamidation (Bidinosti et al., 2010; Deverman et al., 2002), implying that protein deamidation is likely regulated. However, whether protein deamidation is catalyzed by eukaryotic deamidase remains unknown.

RIG-I and MAVS are crucial for containing invading pathogens. Loss of RIG-I or MAVS severely compromises host defense and greatly increases viral replication, as demonstrated by gene knockout studies in mice (Kato et al., 2006; Sun et al., 2006). Viruses have evolved elaborate strategies to evade host antiviral defenses (Ishii et al., 2008). Human hepatitis C virus and related positive-stranded RNA viruses encode conserved proteases that cleave key adaptors (e.g. MAVS and TRIF), effectively terminating innate immune signaling cascades (Foy et al., 2005; Li et al., 2005). Remarkably, our recent studies have shown that murine gamma herpesvirus 68 (γHV68), a model herpesvirus closely-related to human oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), hijacks the MAVS-IKKβ pathway to promote viral lytic replication. Specifically, γHV68 usurps activated IKKβ to degrade RelA, a key subunit of the transcriptionally active NF-κB, thereby negating antiviral cytokine production (Dong and Feng, 2011). In collaborating with the viral RTA E3 ligase (Dong et al., 2012), IKKβ phosphorylates RelA, which primes RelA for the proteasome-mediated degradation.

To dissect innate immune evasion by γHV68, we screened a cDNA library of herpesviral genes and identified the herpesviral vGAT, a homologue of glutamine amidotransferase (GAT) that activates RIG-I. We discovered that vGAT induced RIG-I deamidation and concomitant activation. Although vGAT shares homology with cellular PFAS, vGAT contains no enzymatic activity. However, purified vGAT and PFAS are sufficient to deamidate RIG-I in vitro. Loss of RIG-I and depletion of PFAS resulted in increased inflammatory cytokine production in response to γHV68 infection. Finally, our data demonstrate that γHV68, KSHV and herpes simplex virus 1 (HSV-1) have the ability to induce RIGI deamidation following infection, suggesting that deamidation is a common means to deflect RIG-I-mediated signaling.

RESULTS

RIG-I is Critical for γHV68 to Evade Cytokine Production

We previously reported that murine gamma herpesvirus 68 (γHV68) usurps MAVS and IKKβ to evade cytokine production via inducing RelA degradation that is facilitated by the viral RTA E3 ligase (Figure 1A)(Dong et al., 2012). RIG-I and MDA5 are the two known cytosolic sensors upstream of MAVS. We then examined whether RIG-I and MDA5 are necessary for RelA degradation induced by γHV68 infection. In Rig-i+/+ mouse embryonic fibroblasts (MEFs), γHV68 infection reduced RelA protein at 2 and 4 hours post-infection (hpi) and, by 8 hpi, RelA protein returned to levels of mock-infected cells. In contrast, RelA protein remained constant in γHV68-infected Rig-i−/− MEFs (Figure 1B), phenotypically recapitulating the roles of MAVS in γHV68-induced RelA degradation. “Reconstituted” expression of RIG-I in Rig-i−/− MEFs restored RelA degradation (Figure S1A). Remarkably, the ATPase-deficient RIG-I-K270A (Saito et al., 2007) was as effective as wild-type RIG-I in promoting RelA degradation. Moreover, γHV68 infection reduced RelA protein with similar kinetics in Mda5+/+ and Mda5−/− MEFs (Figure S1B). These results show that RIG-I and MAVS are crucial for γHV68-induced RelA degradation.

Figure 1. RIG-I is critical for γHV68 to evade cytokine production.

Figure 1

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(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-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 Enzyme-linked Immunosorbent Assay (D). **p<0.01, ***p<0.001.

(E) 293T/Flag-RIG-I stable cell line was mock-infected, infected with γHV68 (MOI=5) or Sendai virus (SeV, 100 Unit/ml) for 3 hours (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).

See also Figure S1.

Next, we analyzed the expression of two representative NF-κB-dependent inflammatory cytokines, CCL5 and IL-6. In Rig-i+/+ MEFs, γHV68 infection up-regulated CCL5 and IL-6 mRNA by ~40-60- and ~4-fold, respectively. Strikingly, γHV68 infection in Rig-i−/− MEFs increased CCL5 and IL-6 mRNA by ~80-160-fold and ~10-20-fold (Figure 1C). Loss of RIG-I, as expected, greatly reduced the expression of IL-6 and TNF-α upon infection by Sendai virus (SeV), a prototype RNA virus (Figure S1C). Enzyme-linked immunosorbent assay (ELISA) showed that, when infected with γHV68, Rig-i−/− MEFs secreted ~2-3-fold more IL-6 than Rig-i+/+ MEFs (Figure 1D). Increased secretion of IL-6 and TNF-α was also observed in MEFs deficient in MAVS in response to γHV68 infection (Figure S1D). To determine whether γHV68 infection activates RIG-I, we examined RIG-I oligomerization by gel filtration. In mock-infected cells, RIG-I was eluted in fractions that correspond to the size of ~120-300 kDa, indicative of a monomer or dimer. RIG-I purified from γHV68-infected cells was detected in fractions corresponding to ~440-670 kDa (Figure 1E), which was comparable to the level of oligomerization of RIG-I induced by SeV infection. These results indicate that RIG-I is activated by γHV68 and required to evade host cytokine production.

vGAT Specifically Interacts with RIG-I

To delineate virus-host interactions in γHV68 immune evasion, we took an independent approach to identify viral factor(s) that activates the RIG-I-MAVSIKKβ cascades. Importantly, RIG-I activation in γHV68-infected cells promotes RelA degradation due to the viral RTA E3 ligase that ubiquitinates RelA (Dong et al., 2012), while RIG-I activation in the absence of γHV68 infection results in NF-κB activation. Thus, we employed the NF-κB reporter assay as a surrogate to screen a cDNA library that expresses 70 out of a total of ~80 putative ORFs of γHV68. We discovered that ORF75c was the most potent activator of NF-κB (Figure 2A and S2A). ORF75c shares homology with cellular GAT, thus referred to as vGAT. RTA expression, as expected, potently diminished vGAT-induced NF-κB activation (Figure S2B). Two other GAT homologues of γHV68, ORF75a and ORF75b, failed to activate NF-κB (Figure S2C). These vGAT homologues are packaged in virion particle and are expressed during early and late stages of lytic replication (Gaspar et al., 2008; Song et al., 2005). vGAT expression increased the nuclear NF-κB activity by electrophoresis mobility shift assay (Figure S2D). Moreover, vGAT expression activated IKKβ and over-expression of a kinase-dead mutant of IKKβ or a degradation-resistant IκBα abolished vGAT-induced NF-κB activation in a dose-dependent manner (Figure S2E-G). Loss of MAVS impaired vGAT-induced NF-κB activation (Figure S2H). MAVS can activate both NF-κB and IRF transcription factors. Interestingly, vGAT expression weakly activated IRF3 and the IFN-β promoter (Figure S2I and J). Moreover, γHV68 infection weakly induced the expression of Ifnb, but not that of Isg56 or IRF3 dimerization (Figure S2K and L). These results support the conclusion that vGAT preferentially activates the IKKβ-NF-κB signaling branch.

Figure 2. vGAT interacts with RIG-I.

Figure 2

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(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 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 h. 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 indicated genes. WCLs were precipitated with anti-Flag (RIG-I). Precipitated proteins and WCLs were analyzed by immunoblotting.

(F) GST pulldown, 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.

See also Figure S2.

To identify cellular vGAT-interacting protein(s), we purified vGAT and analyzed proteins co-purified with vGAT by mass spectrometry. This identified RIG-I as the most abundant vGAT-interacting partner (Figure 2B and S2M). Indeed, vGAT co-precipitated with RIG-I from lysates of γHV68-infected cells (Figure 2C). Co-immunoprecipitation (Co-IP) further showed that vGAT interacted with RIG-I, but not MDA5, in transfected 293T cells (Figure S2N). ORF75a and ORF75b failed to interact with RIG-I (Figure S2O and P), which correlates with the lack of NF-κB activation. These results indicate that vGAT specifically interacts with RIG-I.

RIG-I and vGAT are multi-domain proteins, we performed mutational analysis and analyzed vGAT interaction with RIG-I by co-IP. The result indicated that deletion of the tandem CARDs of RIG-I (referred to as RIG-I-N) abolished its interaction with vGAT and RIG-I-N was sufficient to interact with vGAT (Figure 2D). Similarly, co-IP and in vitro GST pulldown assays revealed that the GAT domain of vGAT was sufficient to bind to RIG-I (Figure 2E and F). Thus, the CARDs of RIG-I interact with the GAT domain of vGAT (Figure 2G).

vGAT Directly Activates RIG-I

Considering that vGAT physically interacts with RIG-I, we determined whether vGAT directly activates RIG-I. Knockdown of RIG-I by two pairs of shRNA (Figure 3A) and loss of RIG-I in MEFs abolished NF-κB activation induced by vGAT expression and SeV infection (Figure 3B and S3A), indicating that RIG-I is essential for vGAT-induced NF-κB activation. To monitor RIG-I activation, we examined the ubiquitination, oligomerization, and MAVS interaction of RIG-I. Using the GST-RIG-I-N construct, we found that vGAT expression potently increased the anti-ubiquitin signal when RIG-I-N was precipitated in RIPA buffer (Figure 3C). vGAT-induced ubiquitination of RIG-I-N was abolished by the expression of the K63R ubiquitin mutant, but increased by that of the K48R mutant, supporting the conclusion that the polyubiquitin chains of RIG-I-N are K63-linked (Gack et al., 2007) (Figure S3B). To determine whether vGAT is necessary to induce RIG-I ubiquitination, we have generated vGAT-deficient γHV68 (γHV68.ΔvGAT) in a vGAT-complementing cell line, given that vGAT is essential for γHV68 replication (Gaspar et al., 2008; Song et al., 2005) (Figure S3C). To detect ubiquitination of endogenous RIG-I, we transfected 293T cells with a plasmid expressing Flag-ubiquitin and infected with γHV68 wild-type or γHV68.ΔvGAT. Immunoblot analysis indicated that γHV68 wild-type, but not γHV68.ΔvGAT, induced RIG-I ubiquitination (Figure 3D). These results show that vGAT promotes RIG-I ubiquitination.

Figure 3. vGAT directly activates RIG-I.

Figure 3

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(A and B) 293T cells stably expressing control or RIG-I shRNA were harvested and whole cell lysates (WCLs) were analyzed by immunoblotting (A). RIG-I-knockdown cells were transfected with NF-κB reporter cocktail or infected with Sendai virus (SeV) (100 HAU for 16 h). NF-κB activation was determined by luciferase assay (B).

(C) 293T cells were transfected with plasmids containing 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 h post-transfection, cells were infected with γHV68 wild-type or γHV68ΔvGAT (MOI=10) for 16 h. WCLs in RIPA buffer were precipitated with anti-RIG-I antibody. Precipitated RIG-I and WCLs were analyzed by immunoblotting with indicated antibodies.

(E) 293T cells were transfected with plasmids containing 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, infected with wild-type γHV68 (MOI=10) or γHV68.ΔvGAT for 16 h. Purified RIG-I was analyzed by gel filtration. Aliquots (30 μl) of fractions and WCLs were analyzed by immunoblotting.

See also Figure S3.

Next, we purified RIG-I from transfected 293T cells and analyzed RIG-I oligomerization by gel filtration. RIG-I was eluted in fractions corresponding to ~120-300 kilodalton (kD). In the presence of vGAT, significant amount of RIG-I was eluted in fractions corresponding to 440-670 kDa, indicative of oligomerization (Figure 3E and S3D). The closely-related ORF75b failed to induce RIG-I oligomerization and vGAT did not induce MDA5 oligomerization under the same conditions (Figure S3E). RIG-I from resting cells was eluted in fractions corresponding to ~120-300 kDa, whereas a portion of RIG-I eluted in fractions corresponding to ~440-670 kD in γHV68-infected cells. Compared to wild-type γHV68, γHV68.ΔvGAT failed to induce RIG-I oligomerization (Figure 3F and S3F). Finally, vGAT also induced hetero-dimerization between RIG-I and MAVS in a dose-dependent manner in transfected cells (Figure S3G). These results show that vGAT directly activates RIG-I.

vGAT Expression Induces RIG-I Deamidation

While the GAT domain of vGAT was sufficient to interact with RIG-I, it did not activate NF-κB (Figure S4A), suggesting that interaction is not sufficient to activate RIG-I by vGAT. Considering that vGAT is a homologue of PFAS enzyme, we reasoned that the enzyme activity of GAT was important for vGAT-induced NF-κB activation. Thus, we determined NF-κB activation under conditions of treatment with a GAT inhibitor, 6-Diazo-5-oxo-L-norleucine (DON). DON treatment (5 μM) reduced vGAT-induced NF-κB activation by ~60% (Figure 4A). IKKβ-induced NF-κB activation, however, was not altered by DON. Treatment with DON had no significant effect on SeV-induced NF-κB activation and cell viability (Figure S4B and C). These results suggest that GAT enzyme activity is required for vGAT-induced RIG-I activation.

Figure 4. vGAT induces RIG-I deamidation.

Figure 4

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(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 hours 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. Whole cell lysates (WCLs) were analyzed by two-dimensional 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 Figure S3E and F 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 two-dimensional gel electrophoresis and immunoblotting.

(E) 293T/Flag-RIG-I cells were transfected with a plasmid containing γHV68 (mvGAT) or KSHV (k-vGAT) GAT, WCLs were analyzed by two-dimensional gel electrophoresis.

(F) 293/Flag-RIG-I cells were infected with γHV68 (MOI=20), HSV-1 (MOI=2) for 16 h, or SeV (100 HA unit/ml), KSHV (MOI=10), influenza virus (PR8) (MOI=5) for 2 h. RIG-I deamidation was analyzed similarly as in (E).

See also Figure S4.

GAT converts a neutral glutamine or asparagine to a negatively charged glutamate or aspartate, respectively. We used two-dimensional gel electrophoresis (2-DGE) to assess the charge status of RIG-I. Due to the difficulty of analyzing endogenous RIG-I by 2-DGE, we established 293 cells stably expressing RIG-I. When vGAT was expressed, RIG-I migrated toward the anode side, indicating increased negative charge of RIG-I. vGAT and the robustly expressed ORF75b did not alter the charge status of MDA5 (Figure S4D) and RIG-I (Figure 4B), respectively. We then purified Flag-RIG-I and analyzed peptides for possible deamidation of glutamines and asparagines. Tandem mass spectrometry analysis identified three putative deamidated sites, i.e., glutamine 10 (Q10), asparagine 245 (N245) and 445 (N445) (Figure 4C, S4E and F). We engineered a RIG-I triple deamidation mutant, RIG-I-TD, in which all three residues were replaced with the corresponding deamidated residues (i.e. Q10E, N245D, and N445D). As shown by 2-DGE analysis, RIG-I-TD was mainly detected within a region to the anode side of wild-type RIG-I (Figure 4D). Moreover, vGAT expression caused a migration shift of RIG-I toward the anode to a point similar to RIG-I-TD, and did not affect the migration of RIG-I-TD (Figure 4D). Finally, the migration pattern of RIG-I and RIG-I-TD was not affected by phosphatase treatment, arguing against the contribution of phosphorylation to the migration pattern of RIG-I and RIG-I-TD (Figure S4G). Together, these results indicate that Q10, N245, and N445 are indeed the residues that undergo deamidation upon vGAT expression.

Infection of Herpesviruses Induces RIG-I Deamidation and Activation

The vGAT genes are conserved in all gamma herpesviruses. To determine whether these vGATs have conserved functions, we examined NF-κB activation by reporter assay. vGATs of γHV68 (m-vGAT) and KSHV (k-vGAT), but not that of EBV (e-vGAT), activated NF-κB (Figure S4H). Consistent with this, m-vGAT and k-vGAT induced RIG-I oligomerization by gel filtration (Figure S4I). Next, we examined the effect of vGATs on RIG-I charge by 2-DGE. Similar to m-vGAT, kvGAT expression resulted in robust shift of RIG-I toward the anode, indicative of deamidation (Figure 4E). We further examined RIG-I deamidation after de novo infection of murine γHV68, human KSHV and HSV-1. Infection of these herpes viruses increased negative charge of RIG-I, indicative of deamidation (Figure 4F, S4J and K). Infection of influenza virus or SeV, however, did not induce RIG-I deamidation. These results show that herpesvirus infection or expression of vGATs can induce RIG-I deamidation and concomitant activation.

Deamidation Induces RIG-I Activation

To assess the effect of deamidation on RIG-I activation, we determined RIG-I-dependent signaling by reporter assay for gene expression, immunoblotting for RIG-I ubiquitination and gel filtration for RIG-I oligomerization. Like RIG-I-N, RIGI-TD expression activated the NF-κB and IFN-β promoters in 293T cells and Raw264 mouse macrophage, whereas wild-type RIG-I and the mutant containing D10A, N245/445A (RIG-I-3A) failed to do so (Figure 5A and S5A-C). Exogenous expression of RIG-I-TD also up-regulated gene expression of inflammatory cytokines, including CCL5 and CxCL-10 (Figure 5B). RIG-I-TD expression induced IRF3 dimerization and Ifnb and Isg56 expression, indicative of activation of the IFN signaling cascade (Figure S5D and E). Moreover, RIG-I-TD was highly ubiquitinated in resting cells compared to wild-type RIG-I (Figure 5C). Gel filtration further showed that wild-type RIG-I eluted in fractions corresponding to ~120-300 kDa, whereas significant amount of RIG-I-TD eluted in fractions corresponding to ~440-670 kDa (Figure 5D). To probe the contribution of individual deamidated residues to RIG-I activation, we mutated these residues, either individually or in combination, and examined the ability of these RIG-I mutants to activate NF-κB and IFN-β promoters. We found that RIG-I mutants harboring one or two deamidated residues minimally activated these reporters (Figure S5F), indicating that deamidation of all three residues are required to fully activate RIG-I. Although RIG-I-TD is activated, SeV infection did not further increase IFN induction by RIG-I-TD, suggesting that RIG-I-TD is inert to viral RNA (Figure S5G). These results collectively indicate that triple deamidations are necessary and sufficient to induce RIG-I activation.

Figure 5. The deamidated RIG-I-TD activates innate immune signaling.

Figure 5

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(A) 293T cells were transfected with NF-κB reporter cocktail and plasmids containing indicated genes, and NF-κB activation was determined by luciferase assay.

(B) 293T cells were transfected with plasmids containing wild-type RIG-I or RIGI-TD. Total RNA was extracted at 24 hour 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 (80 nmole) were incubated with [32P]-labeled 5′-triphosphate RNA (150 nM), without or with 200-fold excess of cold 5′-triphosphate RNA. RNA-RIG-I complex was analyzed by polyacrylamide gel electrophoresis and autoradiography (H).

See also Figure S5.

CARD-mediated oligomerization is crucial for RIG-I activation and signaling. RIG-I-TD was much more potent than RIG-I-DD in activating NF-κB and IFNβ reporters, suggesting that the Q10 deamidation is important for RIG-I activation. To examine the effect of Q10E on CARD-mediated oligomerization, we purified RIG-I-N and RIG-I-N-Q10E from bacteria and performed gel filtration analysis. RIG-I-N eluted primarily as a monomer, with a small fraction of oligomers (Figure 5E). Despite of much lower concentration, RIG-I-N-Q10E had significant levels of dimers and oligomers. Thus, the Q10E mutation promotes CARD-mediated oligomerization, consistent with our observation that deamidation activates RIG-I.

While N245 and N445 are 200 amino acids apart in the primary sequence, they are positioned at close proximity to one another in the three-dimensional structure of the RIG-I helicase domain (Figure 5F)(Kowalinski et al., 2011; Luo et al., 2011). N245 is within the region containing the so-called Motif Q (Q247 and K242), which is involved in binding ATP and determining adenine specificity. N445 is located in a short helix within the linker between Helicase domain 1 (Hel1) and Hel2, which directly interacts with Motif Q. N245 and N445 are sandwiched between two negatively charged residues, E249 and E448 (Figure 5F). Deamidating N245 and N445 to aspartate creates a local structure with four net negative charges, which is anticipated to destabilize the structure and diminish binding and hydrolysis of ATP. Under non-activating conditions, RIG-I mutants lacking the N-terminal tandem CARDs display detectable ATPase activity (Saito et al., 2007). Thus, we purified RIG-IΔN and RIG-IΔN with N245/445D mutations (designated RIG-IΔN-DD) and examined their ATPase activity with varying concentrations of ATP. The results show that the KM of RIG-IΔN-DD for ATP is 520 μM, ~3-fold higher than that of RIG-IΔN (138 μM) (Figure S5H). When RIG-I and RIG-I-TD were examined by in vitro ATP hydrolysis, we found that the ATPase activity of wild-type RIG-I was approximately three-fold of that of RIG-ITD. Strikingly, addition of an RNA activator had no detectable effect on ATP hydrolysis of RIG-I-TD, whereas increased that of RIG-I by four-fold (Figure 5G). In vitro RNA-binding further showed that deamidated RIG-I had reduced ability to bind 5′-triphosphate RNA (Figure 5H and S5I). These results demonstrate that, while the deamidation of N245 and N445 contributes to vGAT-induced RIG-I activation, it is not by enhancing the intrinsic ATPase activity of RIG-I, suggesting the existence of a novel activation mechanism.

While vGAT preferentially activates NF-κB, RIG-I-TD activates both NF-κB and IFN induction, suggesting that vGAT inhibits IFN induction downstream of RIG-I. Thus, we examined the effect of vGAT on IFN induction by RIG-I-TD expression. Considering that vGAT activates NF-κB, we used an ISRE promoter to assess its effect on IRF activation. As expected, vGAT diminished ISRE promoter activity and IFN-β gene expression induced by SeV and RIG-I-TD (Figure S5J-L). TRAF3 was implicated in linking MAVS to IRF activation and IFN induction (Belgnaoui et al., 2012; Saha et al., 2006), we thus examined the effect of vGAT on MAVS interaction with IRF3. Co-IP assay showed that vGAT expression reduced the amount of MAVS precipitated by TRAF3 in a dose-dependent manner (Figure S5M). These results show that vGAT induces RIG-I deamidation and disrupts the MAVS-TRAF3 interaction, to avoid IFN induction.

vGAT Interacts with and Recruits Cellular PFAS to Deamidate RIG-I

Herpesviral vGATs share homology with cellular PFAS that catalyzes purine synthesis. However, residues that constitute the catalytic triad essential for amidotransferase activity are not preserved within the GAT domains of vGATs (Figure S6A). Our biochemical and mutational analyses showed that all cysteines within the GAT domain were not essential for vGAT-induced NF-κB activation and RIG-I deamidation, suggesting that vGAT is a pseudo enzyme (Figure S6B, C and data not shown). One notable function of pseudo enzymes is their ability to dimerize with and activate their enzyme-competent counterpart (Adrain and Freeman, 2012; Jura et al., 2009). Moreover, enzyme activity of cellular GATs is predominantly regulated via oligomerization (Huang et al., 2001). We then examined whether vGAT interacts with its cellular counterpart, PFAS. Interaction between vGAT and PFAS was readily detected by co-IP in γHV68-infected MEFs (Figure 6A). Interestingly, γHV68 infection elevated PFAS protein in MEFs, which correlated with a modest increase in PFAS mRNA (Figure S6D and E). Infection of γHV68, but not that of γHV68.ΔvGAT, promoted RIG-I interaction with PFAS (Figure 6B). Co-IP also showed that vGAT interacted with PFAS in transfected 293T cells, and that none of the three structural domains of vGAT was sufficient to interact with PFAS, indicating distinct structural requirement of vGAT for its interaction with PFAS and RIG-I (Figure S6F). vGAT expression in 293T cells increased RIG-I interaction with PFAS in a dose-dependent manner (Figure S6G). Compared to SeV infection, γHV68 infection potently increased RIG-I interaction with PFAS (Figure 6C). These results support the conclusion that vGAT recruits PFAS to RIG-I.

Figure 6. vGAT recruits cellular PFAS to deamidate RIG-I.

Figure 6

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(A and B) MEFs were infected with γHV68 (MOI=5) and whole cell lysates (WCLs) were precipitated with anti-PFAS antibody (A). 293 cells were infected with γHV68 or γHV68.ΔvGAT (MOI=5) for 16 h. 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 unit/ml) for 16 h. 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 hours post infection, cells were harvested and WCLs were analyzed by immunoblotting (D). Transfection of 293T and NF-κB luciferase reporter assay were performed (E). *p<0.05; **p<0.01; ***p<0.001 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 two-dimensional gel electrophoresis and immunoblotting (right).

(F) In vitro deamidation was carried out as in (E) except RIG-I-N was used. Spectral count of the deamidated peptide containing Q10E was shown. Data represents two independent experiments. ND, not detected. WT and ED, wild-type and the enzyme-dead mutant of PFAS.

See also Figure S6.

To probe the roles of PFAS in vGAT-mediated signaling, we depleted PFAS expression with two pairs of shRNA (Figure 6D) and examined vGAT-induced NF-κB activation. We found that knockdown of PFAS reduced vGAT-induced NF-κB activation by ~75% (Figure 6E). Notably, transient knockdown of PFAS did not reduce cell viability (Figure S6H), and addition of hypoxanthine, the end product of purine synthetic pathway, failed to restore vGAT-induced NF-κB activation in PFAS-knockdown cells (Figure S6I). Thus, PFAS, rather than its metabolic end product, is crucial for vGAT-induced NF-κB activation. To test whether PFAS is sufficient to induce RIG-I activation, we over-expressed PFAS and found that PFAS did not significantly activate NF-κB (Figure S6J), supporting the critical role of vGAT in PFAS-mediated RIG-I deamidation. We then determined whether the enzyme activity of PFAS is necessary for NF-κB activation, utilizing an enzyme-dead mutant of PFAS (PFAS-ED), in which the catalytic triad was disrupted with mutations of C1258S, H1297A and E1299A. PFAS ED expression potently inhibited vGAT-induced NF-κB activation and RIG-I oligomerization (Figure 6F and S6K). Thus, the amidotransferase activity of PFAS is necessary for RIG-I deamidation induced by vGAT.

Next, we purified vGAT, PFAS and GST-RIG-I from transfected 293T cells to homogeneity and examine the ability of PFAS and vGAT to deamidate RIG-I in vitro. With 2-DGE, we observed that purified PFAS and vGAT were sufficient to deamidate RIG-I, which was completely inhibited by DON (Figure 6G). PFAS alone or vGAT plus PFAS-ED did not alter the charge status of RIG-I under the same conditions. Moreover, PFAS-ED expression potently inhibited vGAT-induced RIG-I deamidation (Figure S6L). We also used RIG-I-N as a substrate of PFAS and quantified the Q10E-containing peptide by mass spectrometry. The result showed that PFAS, together with vGAT, yielded significant counts of the Q10E-containing peptide, whereas the level of the deamidated peptide by PFAS, vGAT, or vGAT plus PFAS-ED was not detected (Figure 6H). These results show that vGAT and PFAS are necessary and sufficient to deamidate RIG-I.

PFAS-mediated RIG-I Deamidation is Critical for RIG-I Activation to Negate Cytokine Production During γHV68 Infection

PFAS is essential for vGAT-induced NF-κB activation. vGAT and PFAS are sufficient to deamidate RIG-I in vitro. We then examined RIG-I activation by gel filtration, when PFAS was depleted. In 293T cells expressing control or PFAS shRNAs, RIG-I eluted in fractions corresponding to ~120-300 kDa. Strikingly, PFAS depletion diminished the 440-670 kDa oligomers of RIG-I that were induced by vGAT (Figure 7A). Purified from γHV68-infected cells expressing control shRNA, RIG-I was eluted in fractions corresponding to ~440-670 kDa (Figure S7A), which was diminished by PFAS depletion. Moreover, 2-DGE confirmed that PFAS knockdown abolished RIG-I deamidation induced by vGAT (Figure S7B). These results show that PFAS is critical for RIG-I deamidation and concomitant activation induced by vGAT.

Figure 7. PFAS is critical for RIG-I deamidation and activation to negate cytokine production during γHV68 infection.

Figure 7

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(A) Control (CTL) or PFAS shRNA 293T cells 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 (right top). Whole cell lysates (WCLs) were analyzed by immunoblotting (right panels).

(B-E) Rig-i+/+ MEFs stably express control (CTL) or mPFAS shRNA were harvested, 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, we detected two species of RelA in MEFs infected with lentivirus. The larger RelA species was increased in lentivirus-infected MEFs. For figure D, ***p<0.001.

(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.

Given that γHV68 usurps RIG-I to avoid cytokine production, we then determined whether PFAS knockdown impairs RelA degradation and conversely increases cytokine production in response to γHV68 infection. We depleted PFAS with shRNA-mediated knockdown in MEFs (Figure 7B) and examined cytokine production and RelA degradation. γHV68 infection increased CCL-5 and IL-6 mRNA by ~15- and 100-fold in control MEFs, while elevated CCL-5 and IL-6 mRNA by >40-fold and ~300-fold in PFAS-knockdown MEFs (Figure 7C). Furthermore, PFAS-depleted MEFs secreted ~2-fold of IL-6 compared to control MEFs, when infected with γHV68 (Figure 7D). γHV68 infection gradually reduced RelA proteins at 2 and 4 hpi in control MEFs, whereas RelA protein remained constant in γHV68-infected MEFs that PFAS was depleted (Figure 7E). To determine whether RIG-I deamidation is necessary to evade cytokine production, we “reconstituted” RIG-I expression in Rig-i−/− MEFs with wild-type RIG-I or the deamidation-resistant RIG-I-3A mutant (Figure S7C). Gel filtration analysis showed that γHV68 infection did not activate RIG-I-3A, but SeV infection did. Consistent with this observation, RIG-I-3A failed to restore γHV68-induced RelA degradation in Rig-i−/− MEFs (Figure 7F). Thus, RIG-I deamidation by PFAS is necessary for γHV68 to inhibit cytokine production.

DISCUSSION

We report here that herpesvirus proteins induce RIG-I activation via deamidation, unveiling a novel means by which a PRR is activated by viral infection. Remarkably, the deamidation of all three sites are necessary and sufficient to enable RIG-I activation, indicating the critical roles of each residue in activating RIG-I and lending genetic evidence to support their biochemical identification by mass spectrometry analysis. Signature events of RIG-I activation, including oligomerization, ubiquitination and activation of downstream signaling, were observed for the deamidated RIG-I-TD. Notably, viral infection induces more robust RIG-I activation than vGAT expression as analyzed by gel filtration, suggesting other viral factors may contribute to RIG-I activation as well. Nevertheless, these observations support the conclusion that deamidation is an upstream event of RIG-I activation. An unexpected feature of RIG-I activation by deamidation is the reduced RNA-binding and ATPase activity of RIG-I-TD. Our results suggest that deamidation of N245 and N445 imposes crucial conformational change of the helicase domain and CTD, enabling CARD exposure and subsequent oligomerization that is further facilitated by the deamidation of Q10 within the first CARD. Taken together, deamidation of two asparagines within the helicase domain and one glutamine within the first CARD conceivably triggers RIG-I conformational change and concomitant activation. Interestingly, the herpesviral vGATs do not possess intrinsic amidotransferase activity, rather induce RIG-I deamidation via cellular PFAS. To our knowledge, this is the first example whereby a PRR is activated by an enzyme and identifies the first protein-deamidase in higher eukaryotes.

Similar to MAVS and IKKβ (Dong et al., 2010), RIG-I deamidation and activation were usurped by γHV68 to induce RelA degradation and evade antiviral cytokine production (Figure 7G). As such, loss of RIG-I impaired RelA degradation and elevated cytokine production in response to γHV68 infection. Deletion of vGAT in γHV68 genome or knockdown of PFAS impaired RIG-I deamidation and concomitant activation, demonstrating that the cooperation between vGAT and cellular PFAS in initiating RIG-I-dependent immune signaling. Our observation that, in response to γHV68 infection, MEFs deficient in RIG-I or MAVS secrete more inflammatory cytokines than wild-type MEFs suggests that an MAVS- and RIG-I-independent signaling cascade, such as the cGAS-STING pathway (Sun et al., 2013; Wu et al., 2013), operates in MEFs to initiate innate immune response. Nevertheless, these findings support the conclusion that the RIG-I- and MAVS-dependent innate immune signaling events are exploited by γHV68 to negate inflammatory cytokine production (Figure 7G).

Although vGATs are homologues of cellular PFAS, a previous study (Gaspar et al., 2008) and our current work support the corollary that vGATs do not possess intrinsic enzyme activity and therefore are pseudo enzymes. Purified PFAS failed to deamidate RIG-I in vitro and overexpression of PFAS was insufficient to activate RIG-I and NF-κB, whereas addition of vGAT enabled PFAS to deamidate RIG-I. Thus, vGAT potentially activates PFAS, in addition to its “adaptor” function in recruiting PFAS to RIG-I. These results show that PFAS and vGAT constitute a bona fide protein deamidase, representing a new class of mammalian amidotransferase that is capable of deamidating a protein. Conceivably, other cellular GATs may function as protein deamidase as well. Nevertheless, vGAT appears to enable the protein-deamidating ability of PFAS that otherwise deamidates free glutamine, broadening the substrate spectrum of a cellular GAT, a well-appreciated phenomenon known as the substrate ambiguity in enzyme catalysis (Khersonsky and Tawfik, 2010). A striking example of substrate ambiguity is the cellular transglutaminase that demonstrates enzyme activity toward small molecules (e.g., GTP, amino acids), short peptides and large proteins (Facchiano et al., 2006).

Our study suggests that viral pseudo enzymes activate cellular GATs to deamidate RIG-I. Mechanisms of action of these viral factors remain an intriguing area of exploration, while burgeoning number of pseudo enzymes are found to regulate their active kins (Leslie, 2013). A notable precedent is a point mutant of isocitrate dehydrogenase 1 that lost its original enzyme activity, when dimerized with its wild-type, catalyzes the synthesis of a novel onco-metabolite, 2-hydroxyglutarate (Dang et al., 2009; Yan et al., 2009). Similar to vGAT, the kinase-dead ErbB3 serves as an activator of the EGFR kinase via allosteric dimerization (Jura et al., 2009). Future investigation into the action by which herpesvirus vGATs activate PFAS will likely define new mechanism of enzyme regulation. In conclusion, we show that herpesviral homologues of glutamine amidotransferase recruit cellular PFAS to deamidate and activate RIG-I, revealing a new means by which a cytosolic sensor is activated. This study uncovers an unconventional function of PFAS, a cellular metabolic enzyme, in innate immune signaling and identifies the first bona fide protein-deamidase in eukaryotes.

EXPERIMENTAL PROCEDURES

RIG-I Purification and Gel Filtration

Virus-infected 293/RIG-I stable cells or transfected 293T cells were harvested and lysed in cold Triton X-100 buffer (20 mM Tris, pH=7.5, 150 mM NaCl, 1.5 mM MgCl2, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10% glycerol, 0.5 mM EGTA, 0.5% Triton X-100, 1 mM PMSF and 10 μg/ml leupeptin). Centrifuged supernatant was filtered and subjected to incubation with anti-Flag-conjugated agarose for 4-6 hours at 4°C. Agarose was extensively washed and proteins were eluted with Flag peptide at 0.2 mg/ml.

Gel filtration with superose 6 was performed as described previously (Zandi et al., 1997). Briefly, purified proteins (200-300 μl) were loaded to superose 6 column and subjected to gel filtration analysis with Buffer B (20 mM Tris-HCl [pH7.6], 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 20 mM NaF, 20 mM β-glycerophosphate, 1 mM Na3VO4, 2.5 mM metabisulphite [sodium salt], 5 mM benzamidine). Elution was collected in 0.5 ml fractions and aliquots of fractions were analyzed by immunoblotting.

Two-dimensional Gel Electrophoresis

Cells (1×106) were lysed in 150 μl rehydration buffer (8 M Urea, 2% CHAPS, 0.5% IPG Buffer, 0.002% bromophenol blue) by three pulses of sonication and whole cell lysates were centrifuged at 20,000 g for 15 min. Supernatants were loaded to IEF strips for focusing with a program comprising: 20 V, 10 h (rehydration); 100 V, 1 h; 500 V, 1 h; 1000 V, 1 h; 2000 V, 1 h; 4000 V, 1 h; 8000 V, 4h. After IEF, strips were incubated with SDS equilibration buffer (50 mM Tris-HCl [pH8.8], 6 M urea, 30% glycerol, 2% SDS, 0.001% Bromophenol Blue) containing 10 mg/ml DTT for 15 min and that containing 2-iodoacetamide for 15 min. Strips were washed with SDS-PAGE buffer, resolved by SDS-PAGE, and analyzed by immunoblotting.

Additional experimental procedures, including reagents, lentivirus, viral infection, immunoprecipitation and immunoblotting, knockdown, in vitro kinase assay and deamidation assay, can be found in Supplemental Information.

Supplementary Material

ACKNOWLEDGEMENTS

We thank Dr. Takeshi Saito for reading of the manuscript. We thank Drs. Paul Lieberman and Yan Yuan for vGAT plasmid, Micheala Gack for Rig-i−/− MEF, Ting-ting Wu and Ren Sun for γHV68 BAC, Steve Gygi and Ross Tamarin for mass spectrometry analysis. We thank Ms. Stacey Lee for editing of this manuscript. This work is supported by grants from NIH (DE021445 and CA134421) and ACS (RSG-11-162-01-MPC), and core services performed through Norris Cancer Center grant P30CA014089-34.

Footnotes

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SUPPLEMENTAL INFORMATION Supplemental information includes seven figures and Extended Experimental Procedures.

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