In vitro Reconstitution of a RIG-I Signaling Cascade from RNA to IRF3 Activation
To reconstitute the RIG-I pathway in vitro, we first examined whether RIG-I isolated from virus infected cells could cause IRF3 activation in the presence of mitochondria, which contain MAVS, and cytosolic extracts, which contain TBK1 and many other components. A hallmark of IRF3 activation is its dimerization, which depends on its phosphorylation by TBK1 and can be measured by native gel electrophoresis (Yoneyama et al., 2002
). To set up the assay, HEK293T cells stably expressing full-length RIG-I with a C-terminal Flag tag were infected with Sendai virus (SeV) or uninfected, then RIG-I was affinity purified (). Crude mitochondria (P5) and cytosolic extracts (S5) were prepared from uninfected HEK293T cells by differential centrifugation (), and 35
S-labeled IRF3 protein was synthesized by in vitro translation. As shown in , dimerization of IRF3 was observed when RIG-I from virus-infected cells was incubated with mitochondria (P5) and cytosolic extracts (S5) in the presence of ATP (lane 3). In contrast, RIG-I from mock-treated cells did not promote IRF3 dimerization (lane 2). The activation of IRF3 required both cytosol and mitochondria (lanes 5 & 6). Mitochondria isolated from cells depleted of MAVS by RNAi could not support IRF3 dimerization (Supplementary Figure S1A
), confirming that MAVS is essential for activating the downstream pathway in this in vitro assay. When RIG-I was isolated from cells depleted of TRIM25 by RNAi, its ability to promote IRF3 dimerization in the in vitro assay was greatly reduced (Supplementary Figure S1B
), supporting an important role of TRIM25 in RIG-I activation. As we have shown recently, mitochondria isolated from virus-infected cells activated IRF3 in the absence of RIG-I (, lane 1) (Zeng et al., 2009
In Vitro Reconstitution of the RIG-I Pathway and Regulation of RIG-I by RNA- and Ubiquitination
Transfection of cells with synthetic RNAs, such as the dsRNA analogue poly(I:C) and 5′-pppRNA, potently induces IRF3 dimerization. To test if RNA could activate the entire RIG-I pathway in vitro, we expressed and purified full-length RIG-I from insect cells (Sf9; , lower left) and incubated it with ATP and a 79-nucleotide (79nt) 5′-pppRNA (Supplementary Figure S1C
). This RNA strongly induced IFNβ when transfected into HEK293-IFNβ-luciferse reporter cells (Supplementary Figure S1D
). However, incubation of this RNA with the recombinant RIG-I protein did not cause IRF3 dimerization in the reconstituted system (, lane 1 in lower right panel). As ubiquitination has been shown to be important in the RIG-I pathway, we incubated RIG-I with E1, Ubc5c (E2), TRIM25 (E3), and ubiquitin together with ATP and RNA. Remarkably, this condition caused RIG-I to activate the entire pathway, resulting in IRF3 dimerization (, lane 3 in lower right panel). A K270A mutation in the ATPase domain of RIG-I, which abrogates the ability of RIG-I to induce interferons in vivo (Yoneyama et al., 2004
), also abolished its ability to induce IRF3 dimerization in vitro. Poly(I:C) stimulated IRF3 dimerization in this reconstituted system in a manner dependent on ubiquitin, E1, Ubc5c, TRIM25 and RIG-I (). Activation of RIG-I was also observed with another 5′-pppRNA containing 135 nucleotides (135nt) (). Dephosphorylation of the 5′-pppRNAs with shrimp alkaline phosphatase (SAP) destroyed their ability to activate IRF3 in vitro () and IFNβ induction in HEK293T cells (Supplementary Figure S1D
). In contrast, treatment of poly(I:C) with SAP did not inhibit its activity, consistent with the previous report that this dsRNA analogue activates RIG-I and MDA5 in a manner independent of 5′-triphosphate (Kato et al., 2008
). As expected, the DNA poly(dI:dC) did not activate the RIG-I pathway in vitro or in cells ( & Supplementary Figure S1D
). In vitro reconstitution of the RIG-I pathway also led to site-specific phosphorylation of IRF3 and IκBα (Supplemental Results and Figures S1E, F & G
To determine if RIG-I could be activated by naturally occurring viral RNA, we incubated purified RIG-I protein with total RNA from HEK293T cells infected with Sendai virus. Indeed, RNA from virus-infected but not mock-treated cells stimulated RIG-I to activate IRF3 (, lanes 1–3). To estimate the sensitivity of 5′-pppRNA detection by RIG-I, we incubated RIG-I with different amounts of 5′-pppRNA in the presence or absence of HEK293T cellular RNA (, lanes 4–18). The half maximal effective concentration (EC50) of the 5′-pppRNA was estimated at 1.2 nM and 0.4 nM in the absence and presence of the cellular RNA, respectively (). It is not clear how the cellular RNA enhances the potency of 5′-pppRNA, but one possibility is that these RNAs reduce the non-specific loss of very small amounts of 5′-pppRNA in the reactions. In any case, the fact that the presence of a large excess of cellular RNA does not interfere with the specific recognition of 5′-pppRNA by RIG-I underscores the remarkable specificity of viral RNA detection by RIG-I. Assuming that the cytoplasm of a human cell has a volume of ~500 μm3, we estimated that less than 20 molecules of viral RNA in a cell (equivalent to ~0.07 nM) are sufficient to trigger detectable IRF3 dimerization. Thus, our in vitro reconstitution recapitulates the entire RIG-I pathway with exquisite sensitivity and specificity for 5′-pppRNA (see Discussion).
Ubc5 and Ubc13 are Required for the Activation of RIG-I and MAVS
Ubc5 is a family of E2s comprising highly homologous isoforms (Ubc5a, b & c; putative Ubc5d in human) that catalyze the synthesis of polyUb chains linked through various lysines of ubiquitin, including K63 (Xu et al., 2009
). In contrast to Ubc5, the E2 complex consisting of Ubc13 and Uev1A is highly specific in synthesizing K63 polyUb chains (Deng et al., 2000
; Hofmann and Pickart, 1999
). To determine the E2 involved in RIG-I activation, we examined a panel of E2s for their ability to stimulate RIG-I in the presence of TRIM25 and RNA (). The Ubc5 isoforms (Ubc5a, b & c) and Ubc13/Uev1A were capable of stimulating RIG-I to promote IRF3 dimerization. In contrast, Ubc3, Ubc7 and E2-25K had no activity (), despite the ability of these E2s to form thioesters with ubiquitin (data not shown; see also Zeng et al, 2009
). To test if Ubc5 and/or Ubc13 are involved in the activation of MAVS by RIG-I, we established human osteosarcoma U2OS cell lines stably integrated with tetracycline-inducible shRNA vectors targeting both Ubc13 and two isoforms of Ubc5 (Ubc5b & c) (Xu et al., 2009
). As shown in , the mitochondria from the cells depleted of Ubc13, Ubc5b and Ubc5c lost the ability to activate IRF3. These results suggest that Ubc5a and Ubc5d, which are not targeted by the Ubc5 shRNAs, play a minor role in IRF3 activation in U2OS cells. RNAi of Ubc13 or Ubc5 alone partially inhibited the IRF3-stimulatory activity of the mitochondria from virus-infected cells (Supplementary Figure S2A
; Figure S3 in Zeng et al. 2009
). These results suggest that both Ubc5 and Ubc13 are involved in the activation of MAVS in the mitochondria by RIG-I in response to viral infection.
K63 polyubiquitination is Essential for RIG-I Activation
K63 Polyubiquitination is Essential for the Activation of RIG-I and MAVS
To determine if K63 of ubiquitin is required for RIG-I activation in the in vitro system, we incubated various ubiquitin lysine mutants with RIG-I in the presence of RNA, ATP, E1, TRIM25 and Ubc5c or Ubc13/Uev1A. The reaction mixture was then incubated with the mitochondrial fraction (P5) to activate MAVS. The activated mitochondria were isolated and then tested for their ability to stimulate IRF3 dimerization in the presence of cytosolic extracts (). The ubiquitin proteins containing a lysine at position 63 (wild-type, K48R and K63-only) were capable of activating RIG-I, whereas those containing a substitution at K63 (K63R, K48-only, KO and methylated ubiquitin) had no activity (; see Supplementary Figure S2C
for an illustration of ubiquitin mutants). Interestingly, although Ubc5c and TRIM25 catalyze the synthesis of polyUb chains from K63R (Supplementary Figure S2D
, lane 2), these chains did not stimulate RIG-I (, lane 2), indicating that K63 polyUb chain synthesis is specifically required for RIG-I activation in vitro.
To investigate the role of K63 polyubiquitination in the activation of MAVS in cells, we used recently developed U2OS cell lines in which endogenous ubiquitin is replaced with wild-type or K63R mutant of ubiquitin through a tetracycline-inducible strategy (Xu et al., 2009
). As shown in , depletion of endogenous ubiquitin severely impaired the ability of the mitochondria to promote IRF3 dimerization, but this activity was rescued by the expression of the wild-type ubiquitin transgene. In contrast, when the endogenous ubiquitin was replaced with the K63R mutant, the mitochondria isolated from these cells failed to activate IRF3 in the vitro assay, strongly suggesting that K63 polyubiquitination is essential for viral activation of MAVS in the mitochondria.
K63 Polyubiquitin Chains Activate RIG-I through its N-terminal CARD Domains
The N-terminus of RIG-I contains tandem CARD domains, which, when overexpressed in mammalian cells, constitutively activate IRF3 (Yoneyama et al., 2004
). To determine if this N-terminal fragment [RIG-I(N), see ] could activate IRF3 in vitro, we expressed the protein in E. coli
and purified it to near homogeneity (). When the protein was incubated with mitochondria and cytosolic extracts, it did not promote IRF3 dimerization (, lane 2). However, when the incubation mixtures contained ubiquitination components, including E1, Ubc5c, TRIM25 and ubiquitin, robust IRF3 dimerization was detected.
Activation of RIG-I N-terminus by K63 Polyubiquitin Chains
To determine if ubiquitination of RIG-I(N) is required for IRF3 activation in the in vitro system, we carried out a ubiquitination reaction in the presence of different pairs of E2 and E3, and then treated the reaction mixture with the chemical N-ethylmaleimide (NEM), which alkylates the active site cysteine of E1 and E2. The reaction mixtures were then incubated with GST-RIG-I(N) before further incubation with the mitochondrial and cytosolic fractions to measure IRF3 dimerization (). Under these conditions, RIG-I(N) was not ubiquitinated because E1 and E2 had been inactivated by NEM. Remarkably, when TRIM25 or another RING domain E3 TRAF6 was incubated with either Ubc13/Uev1A or Ubc5c, robust IRF3 dimerization was detected (lanes 2–5). When K48 polyUb chains were synthesized by Ubc3 and an E3 complex consisting of Skp1, Cul-1, Roc1 and βTrCP2 (SCF-βTrCP2), these chains did not activate the RIG-I pathway. Similarly, linear polyUb chains generated by Ubc5c and an E3 complex consisting of HOIP-1 and HOIL-1L (termed LUBAC) led to very weak IRF3 dimerization. To further test if ubiquitin chains of other linkages could support IRF3 dimerization, we used a panel of ubiquitin mutants harboring a single lysine (). Although all the ubiquitin mutants were capable of forming polyUb chains in the presence of Ubc5c and TRIM25, the only mutants capable of supporting IRF3 activation were those containing a lysine at position 63 (K63-only and His6
-K63 only). Collectively, these results clearly demonstrate that polyUb chains containing the K63 linkage, but not other linkages, specifically activate the RIG-I pathway. We also found that unanchored polyUb chains, but not ubiquitinated TRIM25, mediate RIG-I activation (Supplementary Results and Figure S3
Short, Unanchored, K63-Linked Ubiquitin Chains Activate RIG-I
To determine if short ubiquitin chains are capable of activating RIG-I, we incubated GST-RIG-I(N) with a ubiquitin polymer containing four units of ubiquitin linked through K63 (K63-Ub; ). Strikingly, incubation of GST-RIG-I(N) with K63-Ub4 led to robust activation of IRF3 in this assay (lane 2), whereas GST-RIG-I(N) or K63-Ub4 alone had no activity (lanes 1 & 3). We then tested a panel of ubiquitin chains of different lengths and linkages for their ability to activate RIG-I(N) (). Interestingly, K63 Ub chains containing more than two ubiquitin moieties potently activated RIG-I(N) (, lanes 3–8). K63-Ub2 had very weak activity (lane 2), whereas monomeric ubiquitin and K48-linked ubiquitin chains were inactive (lane 1; lanes 11–14). Ub4 containing mixed linkages of K48 and K63 also had greatly reduced activity (lanes 9–10). We then carried out titration experiments to quantify the relative potency of different ubiquitin chains in the activation of RIG-I(N) (). Similar titration experiments were also carried out using full-length RIG-I in the presence of 5′-pppRNA (). Among the ubiquitin chains tested, K63-Ub4 was the most potent activator, with an EC50 of ~25 nM for RIG-I(N) activation. The potency of K63-Ub3 was about 27 fold less (EC50 ≈ 680 nM), whereas K63-Ub2 was nearly inactive even at the highest concentration (1 μM). Ubiquitin, K48-Ub3 and linear-Ub3 were inactive at all the concentrations tested. These results demonstrate that short, unanchored K63 ubiquitin chains containing at least three ubiquitin moieties are potent and specific activators of RIG-I.
Short, Unanchored, K63-Ubiquitin Chains Potently Activate RIG-I
The Tandem CARD Domains of RIG-I Bind to K63 Ubiquitin Chains
The potent activation of RIG-I(N) by K63 ubiquitin chains implies that the N-terminus of RIG-I binds to these chains. Indeed, GST-RIG-I(N) was able to pull down K63-Ub3, but not K48-Ub3 or linear Ub3 (). Both CARD domains are required for ubiquitin binding, because the fragments containing residues 11–200 or 2–180 of RIG-I failed to bind K63-Ub4 or activate IRF3 (; see also Supplementary Figure S5A & S5B
). We also generated a RIG-I(N) protein containing a T55I mutation, which was previously shown to render RIG-I defective in interferon induction (Sumpter et al., 2005
). Interestingly, T55I mutation severely impaired the ability of RIG-I(N) to bind K63-Ub4 and activate IRF3 (, lane 10), suggesting that the signaling defect of this mutant may be due to its failure to bind K63 ubiquitin chains.
RIG-I CARD Domains Bind K63 Ubiquitin Chains and This Binding in Full-Length RIG-I is Regulated by RNA and ATP
To determine if ubiquitin chain binding induces oligomerization of RIG-I(N), we incubated RIG-I(N) with K63-Ub4, and then performed gel filtration analysis using Superdex-200. K63-Ub4 and RIG-I(N) have predicted molecular masses of ~30 and ~24 kDa, respectively; however, they formed an active complex with an apparent molecular mass of approximately 200 kDa (). When each protein alone was fractionated on the same column, K63-Ub4 and RIG-I(N) eluted at positions corresponding to approximately 25 and 50 kDa, respectively. The elution profile of RIG-I(N) suggests that it forms a dimer, and the binding of K63-Ub4 to RIG-I(N) apparently promotes the formation of an oligomeric complex (e.g, a tetramer).
Full-length RIG-I Binds to K63 Ubiquitin Chains in a Manner that Depends on RNA and ATP
As full-length RIG-I is regulated by RNA binding and ATP hydrolysis, we tested its binding to K63 ubiquitin chains in the presence or absence of RNA and ATP. Immunoprecipitation of His8-RIG-I-Flag led to co-precipitation of K63-Ub4 and long K63 polyUb chains in the presence of 5′-pppRNA and ATP (, lanes 2 & 6). This binding was lost when the RNA was absent or when EDTA was added to chelate Mg2+, which is required for ATP binding. Two different mutations that abrogate the ATPase activity of RIG-I, K270A and D372N, also disrupted the ability of RIG-I to bind K63 polyUb (). To determine if RIG-I binds to RNA and K63 polyUb in a sequential manner, we incubated RIG-I with 5′-pppRNA or K63 polyUb in reciprocal orders (). If RIG-I was pre-incubated with 5′-pppRNA in the first step and then with K63 polyUb in the second step, it was capable of activating IRF3 dimerization in the reconstitution assay (lane 4). In contrast, if RIG-I was pre-incubated with K63 polyUb first, then immunoprecipitated to remove unbound polyUb, its subsequent incubation with 5′-pppRNA failed to activate it (lane 6). Thus, the binding of RIG-I to RNA precedes its binding to K63 polyUb.
Binding of K63 Ubiquitin Chains is Necessary for RIG-I Activation
Previous studies have shown that RIG-I is ubiquitinated at K172 and that a mutation of this residue (K172R) impairs its ability to induce IFNβ (Gack et al., 2007
). Consistent with an important role of K172 in RIG-I activation, we found that GST-RIG-I(N) containing the K172R mutation failed to activate IRF3 in the in vitro reconstitution assay (, lanes 4–6). A GST-RIG-I(N) protein containing mutations at six lysines (K99, 169, 172, 181, 190, 193; herein referred to as 6KR) was also inactive (lanes 10–12), but this activity was rescued by keeping the lysine at position 172 (K172-only; lanes 7–9). Interestingly, the K172R, 6KR and T55I mutants were greatly compromised in their ability to bind K63 polyUb, whereas K172-only was fully capable of binding to these chains (). These results show that the IRF3-activating function of RIG-I correlates well with its ubiquitin binding activity.
Polyubiquitin Binding is Required for RIG-I Activation
To determine if ubiquitination or ubiquitin-binding of RIG-I is important for its activation in cells, we expressed GST-RIG-I(N) and various mutants in HEK293-IFNβ-luciferase reporter cells (), then pulled down these proteins with glutathione-Sepharose (). To distinguish ubiquitin chains covalently conjugated to RIG-I from those non-covalently associated with RIG-I, the Sepharose beads were washed with either phosphate-buffered saline (PBS) or a more stringent buffer containing 0.1% SDS and 1% deoxycholate (RIPA; ). Immunoblotting with a GST antibody showed that while the wild-type GST-RIG-I(N) was conjugated by ubiquitin chains, no apparent ubiquitination of the mutants, including K172R and K172-only, was detected (, lower panel). Immunoblotting of the pull-down proteins with a ubiquitin antibody showed an interesting difference depending on the wash buffers (upper panel). While GST-RIG-I(N) containing K172-only was able to pull down polyUb chains from the cells when the beads were washed with PBS (lane 9), these chains were largely washed away with the RIPA buffer (lane 4), indicating that this mutant was defective in ubiquitination, but retained the ability to bind polyUb chains. Because GST-RIG-I(N)-K172-only was active in inducing IFNβ (), these results suggest that polyUb binding by RIG-I is responsible for its function. The other RIG-I mutants, including K172R, 6KR and T55I, were unable to bind ubiquitin chains in cells, consistent with their inability to induce IFNβ.
To further evaluate whether RIG-I ubiquitination is required for its function, we treated GST-RIG-I(N) isolated from HEK293T cells with a deubiquitination enzyme (DUB), which contains the OTU domain of the Crimean Congo hemorrhagic fever virus (CCHFV) large (L) protein () (Frias-Staheli et al., 2007
; Xia et al., 2009
). The viral OTU (vOTU) can cleave polyUb chains from target proteins as well as unanchored polyUb chains. In contrast, another DUB, isopeptidase T (IsoT) only cleaves unanchored polyUb chains (Reyes-Turcu et al., 2006
). Following treatment with vOTU or IsoT, GST-RIG-I(N) was tested for its ability to activate IRF3 in the in vitro reconstitution assay. Unlike GST-RIG-I(N) expressed in E. coli
, which does not have the ubiquitin system, the same protein expressed in HEK293T cells was able to activate IRF3 without preincubation with ubiquitin chains (, bottom panel), probably because RIG-I(N) isolated from human cells is already bound to endogenous polyUb chains (e.g
., lane 9 in ). Interestingly, although vOTU removed most of the ubiquitin chains from GST-RIG-I(N) (, lanes 6 & 9 in upper panel), the deubiquitinated RIG-I protein was as active in causing IRF3 dimerization as the protein that was mock treated (lanes 1–4 and 9–12, lower panel). These results suggest that ubiquitination of the RIG-I CARD domains may be dispensable for its activity.
Unanchored K63 Polyubiquitin Chains Isolated from Human Cells are Potent Activators of RIG-I
In , we noted that IsoT treatment did not remove much of the polyUb chains from GST-RIG-I(N) (lane 5 in upper panel), indicating that RIG-I(N) protects the ubiquitin chains from degradation by IsoT (see Supplemental Results and Figure S6
). The protection of polyUb by RIG-I(N) offers an opportunity to detect the otherwise labile ubiquitin chains in cells, but poses another challenge of how to release these chains while preserving their functions. We devised a protocol to isolate the RIG-I-bound ubiquitin chains from HEK293T cells by employing two strategies (). First, we lysed the cells in the presence of NEM to inactivate most of the deubiquitination enzymes. Ubiquitin contains no cysteine, therefore evading modification by NEM. Second, as ubiquitin is known to be relatively stable at high temperatures, we heated the RIG-I(N):polyUb complex at different temperatures to identify a condition that denatures RIG-I(N) but preserves the structure and function of ubiquitin chains. Following centrifugation that precipitates denatured protein aggregates, the supernatant is expected to contain polyUb chains and can be tested for its ability to activate IRF3 in the presence of fresh RIG-I(N). Because GST-RIG-I(N)-K172-only appears to capture more polyUb chains, we expressed this protein in HEK293T cells (). In control experiments, we incubated GST-RIG-I(N)-K172-only protein with K63 polyUb chains in vitro, carried out GST pull-down and then heated the complex (lanes 1–6). Five minutes of heating at 70–80°C was sufficient to inactivate the RIG-I protein (lane 1, 3, 5) and release polyUb chains into the supernatant, which were capable of activating IRF3 when supplied with fresh GST-RIG-I(N) (lane 2, 4, 6). When the GST-RIG-I(N) protein expressed in HEK293T cells was heated at 70°C, it was still capable of activating IRF3 in the absence of fresh RIG-I(N) (lane 7), indicating that the activated RIG-I(N) complex was resistant to NEM and 70°C treatment. However, raising the temperatures to 75°C and 80°C inactivated the GST-RIG-I(N) protein (lanes 9 & 11). Strikingly, the supernatant containing polyUb chains released from the RIG-I(N) complex at the high temperatures were capable of activating IRF3 when supplied with fresh GST-RIG-I(N) (lanes 10 & 12).
Endogenous Unanchored K63 Polyubiquitin Chains Activate the RIG-I Pathway
To determine whether the supernatant of the heated GST-RIG-I(N) complex contained unanchored polyUb chains and whether these chains were responsible for activating the RIG-I pathway, we performed two sets of experiments. First, we incubated the supernatant with E1 to determine if the endogenous ubiquitin chains could form thioesters with E1. Indeed, in the presence of E1 and ATP, substantial fractions of both synthetic and endogenous ubiquitin chains formed thioesters that were sensitive to reduction by β-mercaptoethanol, indicating that they contained unanchored C-termini (Supplementary Figure S7A
). Second, we incubated the endogenous ubiquitin chains with IsoT and then measured their activity in the IRF3 dimerization assay (). Importantly, the IsoT treatment completely abolished the ability of the supernatant to activate IRF3 in the presence of GST-RIG-I(N) (lane 8). However, if we reversed the order by incubating the supernatant with GST-RIG-I(N) first and then with IsoT, IRF3 dimerization was observed (lane 9). Parallel control experiments show that unanchored K63 polyUb chains were sensitive to IsoT treatment but became resistant after pre-incubation with GST-RIG-I(N) (lanes 2–5). To directly visualize unanchored polyUb chains associated with the GST-RIG-I(N) complex isolated from HEK293T cells, the heated supernatants treated with and without IsoT were analyzed by immunoblotting with a ubiquitin antibody (, right panel). Several bands in the range of 40–75 kDa, most prominently the 40 kDa band, disappeared in the IsoT treated sample, indicating that these are unanchored ubiquitin polymers containing approximately 5–10 ubiquitin moieties. However, most of the high molecular weight bands remained resistant to IsoT treatment, suggesting that they represent ubiquitin chains conjugated to some protein targets. Because the IsoT treatment completely abolished the activity of the heat supernatant to promote IRF3 dimerization (, lane 8 on left panel), the high molecular weight ubiquitin conjugates remaining after IsoT treatment apparently did not contribute to RIG-I activation.
To determine if the endogenous unanchored polyUb chains are linked through K63 of ubiquitin, we treated the heat-resistant supernatant with the K63-specific deubiquitination enzyme CYLD. This treatment markedly diminished the ability of the supernatant to activate RIG-I (, left panel). CYLD also destroyed the ubiquitin chains with molecular masses in the range of 40–75 kDa, which could be detected with an antibody specific for the K63 ubiquitin linkage (, right panel).
TRIM25 and CYLD have previously been shown to be important for the activation and inactivation of the RIG-I pathway, respectively (Friedman et al., 2008
; Gack et al., 2007
; Zhang et al., 2008
). To determine if these enzymes are involved in the synthesis and disassembly of endogenous K63 ubiquitin chains, we used siRNA to knock down the expression of TRIM25 or CYLD in HEK293T cells, then isolated the endogenous ubiquitin chains as outlined in . Immunoblotting experiments showed that depletion of TRIM25 diminished, whereas depletion of CYLD enhanced, the production of endogenous polyUb chains, which in turn activated IRF3 ().
To evaluate the potency of endogenous polyUb chains in activating RIG-I, we carried out titration experiments using different amounts of heat-resistant supernatants isolated from HEK293T cells (). The amount of endogenous K63-Ub6 was estimated by comparing to synthetic K63-Ub6 using semi-quantitative immunoblotting (Supplementary Figure S7B
). Because K63-Ub6 is the dominant species of endogenous polyUb chains, its concentration was used to calculate the EC50 for stimulating RIG-I. Remarkably, the EC50 for the endogenous ubiquitin chains was estimated to be approximately 50 picomolar (pM), indicating that these ubiquitin chains are highly potent ligands of RIG-I.
Finally, we examined whether viral infection induces the formation of polyUb chains associated with full-length RIG-I in cells (Supplementary Figure S7C
). HEK293T cells expressing RIG-I-Flag were infected with Sendai virus or mock treated, and then the RIG-I complex was immunoprecipitated and heated at 76°C to release polyUb chains. The heat supernatant prepared from the virus-infected cells, but not mock-treated cells, stimulated IRF3 dimerization in the presence of RIG-I(N). This activity was diminished by IsoT treatment (Supplementary Figure S7D
), indicating that the supernatant contained unanchored polyUb chains. It should be noted that the activity associated with full-length RIG-I was much lower than that associated with RIG-I(N), because the activation of full-length RIG-I is limited by its accessibility to viral RNA (see Discussion). Nevertheless, given the potency of 5′-ppp-RNA and unanchored polyUb chains in activating RIG-I, small amounts of viral RNA and endogenous ubiquitin chains are likely to be sufficient to trigger the RIG-I signaling cascade.
In sum, the results presented herein demonstrate that: a) the unanchored K63 ubiquitin chains are present in human cells; b) they are bound and protected by RIG-I; c) they are potent activators of the RIG-I pathway. We propose that sequential binding of RIG-I to RNA and unanchored K63 polyUb chains leads to full activation of RIG-I, which in turn activates MAVS and downstream signaling cascades ().