figure 1. in vitro reconstitution of the rig-i pathway and...

Figure 1. In Vitro Reconstitution of the RIG-I Pathway and Regulation of RIG-I by RNA and Ubiquitination

(A) Purification of RIG-I protein from Sendai virus-infected (+SeV) or untreated (�SeV) HEK293T cells stably expressing RIG-I containing a C-terminal Flag

epitope.

316 Cell 141, 315–330, April 16, 2010 ª2010 Elsevier Inc.

To dissect the signaling mechanisms in the RIG-I pathway, we

establish a cell-free system that mimics viral infection in intact

cells. This in vitro reconstitution system contains recombinant

RIG-I protein, mitochondria, cytosol, RNA, and ubiquitination

enzymes including TRIM25. We demonstrate that 50-pppRNA

and dsRNA, which resemble viral RNA, can activate the entire

RIG-I signaling cascade in vitro, leading to activation of IRF3

and NF-kB. Interestingly, we find that upon incubation with

both RNA and unanchored K63-polyubiquitin (polyUb) chains,

RIG-I is fully activated to cause IRF3 dimerization. We show

that the N terminus of RIG-I, termed RIG-I(N), which contains

tandem CARDs, binds specifically to K63-polyUb chains, and

that this binding is both necessary and sufficient to activate

RIG-I(N). Furthermore, we devise a protocol to isolate endoge-

nous ubiquitin chains associated with RIG-I in a human cell line

and demonstrate that these are unanchored K63-polyUb chains

with potent ability to activate RIG-I. Our results support a model

in which RIG-I binds to two ligands, viral RNA and endogenous

ubiquitin chains, in a sequential manner through the C-terminal

RD and N-terminal CARDs, respectively. The binding of both

ligands is required for full activation of RIG-I.

RESULTS

In Vitro Reconstitution of a RIG-I Signaling Cascadefrom RNA to IRF3 ActivationTo 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 dimeriza-

tion, 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 (Figure 1A).

Crude mitochondria (P5) and cytosolic extracts (S5) were

prepared from uninfected HEK293T cells by differential centrifu-

gation (Figure 1B), and 35S-labeled IRF3 protein was synthesized

by in vitro translation. As shown in Figure 1C, dimerization of

IRF3 was observed when RIG-I from virus-infected cells was

(B) Procedures for isolation of crude mitochondria (P5) and cytosol (S5) by differ

(C) Virus-activated RIG-I induced IRF3 dimerization in vitro. The reconstitution

untreated cells, cytosolic extracts (S5) from uninfected cells, 35S-IRF3, and ATP.

(D) In vitro activation of RIG-I by 50-pppRNA and ubiquitination. His6-tagged RIG

(lower left panel), then incubated with 50-pppRNA (79 nucleotides), ATP, and ub

the reaction mixtures were further incubated with mitochondria (P5) and cytosol (S

ization was analyzed by native gel electrophoresis.

(E) In vitro activation of RIG-I by poly(I:C) and ubiquitination. Similar to (D), excep

enzymes was tested.

(F) The role of 50-triphosphate for RNA to activate RIG-I in vitro. Similar to (D), exc

(SAP).

(G) 50-pppRNA and viral RNA are potent activators of the RIG-I pathway. Total RN

cells, then incubated with RIG-I as in (D), followed by IRF3 dimerization assay (lan

amounts of the RNA (135 nt) (0.07 to 70 nM, at 3-fold increments) were incuba

uninfected cells (lanes 5–11), then IRF3 dimerization assay was performed.

(H) IRF3 dimer shown in (G) was quantified with ImageQuant, then plotted again

See also Figure S1.

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 and 6). Mitochondria isolated from cells depleted of

MAVS by RNAi could not support IRF3 dimerization (Figure S1A

available online), 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 (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 (Figure 1C, lane 1) (Zeng et al., 2009).

Transfection of cells with synthetic RNAs, such as the dsRNA

analog poly(I:C) and 50-pppRNA, potently induces IRF3 dimeriza-

tion. 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;

Figure 1D, lower left) and incubated it with ATP and a 79 nucleo-

tide (79 nt) 50-pppRNA (Figure S1C). This RNA strongly induced

IFN-b when transfected into HEK293-IFN-b-luciferse reporter

cells (Figure S1D). However, incubation of this RNA with the

recombinant RIG-I protein did not cause IRF3 dimerization in

the reconstituted system (Figure 1D, 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 (Figure 1D, lane 3 in lower right panel).

A K270A mutation in the ATPase domain of RIG-I, which abro-

gates 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 reconsti-

tuted system in a manner dependent on ubiquitin, E1, Ubc5c,

TRIM25, and RIG-I (Figure 1E). Activation of RIG-I was also

observed with another 50-pppRNA containing 135 nucleotides

(135 nt) (Figure 1F). Dephosphorylation of the 50-pppRNAs with

shrimp alkaline phosphatase (SAP) destroyed their ability to acti-

vate IRF3 in vitro (Figure 1F) and IFN-b induction in HEK293T cells

(Figure S1D). In contrast, treatment of poly(I:C) with SAP did not

inhibit its activity, consistent with the previous report that this

ential centrifugation.

reaction contained mitochondria (P5), RIG-I isolated from virus-infected or

Dimerization of IRF3 was analyzed by native gel electrophoresis.

-I (wild-type or WT) or its ATPase mutant (K270A) was purified from Sf9 cells

iquitination enzymes as outlined in the diagram. After incubation, aliquots of

5) from uninfected cells together with 35S-IRF3 and ATP, and then IRF3 dimer-

t that poly(I:C) was used and the dependency on ubiquitin and ubiquitination

ept that the RNA was pretreated with or without shrimp alkaline phosphatase

A was extracted from HEK293T cells from viral-infected or untreated HEK293T

es 1–3). To measure the potency of 50-pppRNA in RIG-I activation, increasing

ted with RIG-I in the presence (lanes 12–18) or absence of cellular RNA from

st the concentration of 50-pppRNA.

Cell 141, 315–330, April 16, 2010 ª2010 Elsevier Inc. 317

Figure 2. K63 Polyubiquitination Is Essential for RIG-I Activation

(A) Ubc5 and Ubc13/Uev1a activate RIG-I in vitro. RIG-I was incubated with E1, different E2s as indicated, TRIM25, ubiquitin, RNA, and ATP, followed by IRF3

dimerization assay as described in Figure 1D. The E2 proteins (2 mg) were analyzed by Coomassie blue staining (lower panel).

(B) Ubc5 and Ubc13 are required for viral activation of MAVS in the mitochondria. U2OS cells stably integrated with tetracycline-inducible shRNA against Ubc5b/c

and Ubc13 were treated with or without tetracycline (Tet). After viral infection for the indicated time, mitochondrial fraction (P5) was prepared and the MAVS

activity was measured by IRF3 dimerization assay.


dsRNA analog activates RIG-I and MDA5 in a manner indepen-

dent of 50-triphosphate (Kato et al., 2008). As expected, the

DNA poly(dI:dC) did not activate the RIG-I pathway in vitro or in

cells (Figure 1F and Figure S1D). In vitro reconstitution of the

RIG-I pathway also led to site-specific phosphorylation of IRF3

and IkBa (Extended Results and Figures S1E–S1G).

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 (Figure 1G, lanes 1–3). To estimate the sensitivity

of 50-pppRNA detection by RIG-I, we incubated RIG-I with

different amounts of 50-pppRNA in the presence or absence of

HEK293T cellular RNA (Figure 1G, lanes 4–18). The half-maximal

effective concentration (EC50) of the 50-pppRNA was estimated

at 1.2 nM and 0.4 nM in the absence and presence of the cellular

RNA, respectively (Figure 1H). It is not clear how the cellular RNA

enhances the potency of 50-pppRNA, but one possibility is that

these RNAs reduce the nonspecific loss of very small amounts

of 50-pppRNA in the reactions. In any case, the fact that the pres-

ence of a large excess of cellular RNA does not interfere with the

specific recognition of 50-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 mm3, 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 50-pppRNA (see Discussion).

Ubc5 and Ubc13 Are Required for the Activationof RIG-I and MAVSUbc5 is a family of E2s comprising highly homologous isoforms

(Ubc5a, b, and 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 activa-

tion, we examined a panel of E2s for their ability to stimulate

RIG-I in the presence of TRIM25 and RNA (Figure 2A). The

Ubc5 isoforms (Ubc5a, b, and c) and Ubc13/Uev1A were

capable of stimulating RIG-I to promote IRF3 dimerization. In

contrast, Ubc3, Ubc7, and E2-25K had no activity (Figure 2A),

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

(C) K63 of ubiquitin is essential for RIG-I activation in vitro. RIG-I and IRF3 activati

panel) as the E2 and various ubiquitin mutants as indicated. KO, lysine-less mut

(D) K63 polyubiquitination is essential for viral activation of MAVS in the mitocho

endogenous ubiquitin genes and a rescue expression vector for wild-type or K63

After infection by Sendai virus, mitochondria (P5) were prepared to measure MAV

blotting (lower panels). The HA antibody detects HA-Ub(WT) or HA-Ub(K63R) ex

See also Figure S2.

both Ubc13 and two isoforms of Ubc5 (Ubc5b and c) (Xu et al.,

2009). As shown in Figure 2B, 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 acti-

vation in U2OS cells. RNAi of Ubc13 or Ubc5 alone partially in-

hibited the IRF3-stimulatory activity of the mitochondria from

virus-infected cells (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 the Activationof RIG-I and MAVSTo 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

(Figure 2C). 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 (Fig-

ure 2C; see Figure S2C for an illustration of ubiquitin mutants).

Interestingly, although Ubc5c and TRIM25 catalyze the synthesis

of polyUb chains from K63R (Figure S2D, lane 2), these chains did

not stimulate RIG-I (Figure 2C, 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 activa-

tion 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 Figure 2D, depletion of

endogenous ubiquitin severely impaired the ability of the mito-

chondria 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 in vitro assay, strongly suggesting

that K63 polyubiquitination is essential for viral activation of

MAVS in the mitochondria.

K63-Polyubiquitin Chains Activate RIG-I throughIts N-terminal CARDsThe N terminus of RIG-I contains tandem CARDs, which, when

overexpressed in mammalian cells, constitutively activate IRF3

(Yoneyama et al., 2004). To determine if this N-terminal fragment

on assays were performed using Ubc13/Uev1a (upper panel) or Ubc5c (middle

ant; MeUb, methylated ubiquitin.

ndria. U2OS cells stably integrated with tetracycline-inducible shRNA against

R mutant of ubiquitin were grown in the presence or absence of tetracycline.

S activation. The expression of MAVS and ubiquitin was analyzed by immuno-

pressed from the transgene rescue vector.


Figure 3. Activation of RIG-I N Terminus

by K63-Polyubiquitin Chains

(A) Depiction of RIG-I functional domains.

(B)Purification of recombinant GST-RIG-I(N) protein

from E. coli.

(C) Activation of RIG-I(N) by ubiquitination. GST or

GST-RIG-I(N) was incubated with ubiquitination

reaction (E1, Ubc5, TRIM25, and ubiquitin) and/or

poly(I:C) as indicated, then an aliquot of the reac-

tion was further incubated with mitochondria (P5)

and cytosol (S5) to measure IRF3 dimerization.

(D) K63-polyUb chain synthesis, but not ubiquiti-

nation of RIG-I(N), is required for RIG-I activation

in vitro. Ubiquitination reactions containing dif-

ferent combinations of E2s and E3s as indicated

were carried out before N-ethylmaleimide (NEM)

was added to inactivate E1 and E2s. An aliquot

of the reaction mixture was incubated with GST-

RIG-I(N), then further incubated with mitochondria

(P5) and cytosol (S5) to measure IRF3 dimeriza-

tion. Another aliquot of the reaction was analyzed

by immunoblotting with a ubiquitin antibody (lower

panel). SCFb-TrCP2: an E3 complex containing

Skp1, Cul1, Rbx1, and b-TrCP2; LUBAC: an E3

complex containing HOIL-1L and HOIP.

(E) Similar to (D), except that ubiquitination

reactions were carried out in the presence of

Ubc5c, TRIM25, and various ubiquitin mutants

as indicated.

See also Figure S3.

[RIG-I(N), see Figure 3A] could activate IRF3 in vitro, we ex-

pressed the protein in E. coli and purified it to near

homogeneity (Figure 3B). When the protein was incubated with

mitochondria and cytosolic extracts, it did not promote IRF3

dimerization (Figure 3C, lane 2). However, when the incubation

mixtures contained ubiquitination components, including E1,


Ubc5c, TRIM25, and ubiquitin, robust

IRF3 dimerization was detected even in

the absence of RNA (lane 3).

To determine if ubiquitination of RIG-

I(N) is required for IRF3 activation in the

in vitro system, we carried out a ubiquiti-

nation 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 (Figure 3D). Under these

conditions, RIG-I(N) was not ubiquiti-

nated because E1 and E2 had been inac-

tivated 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, Rbx1, and bTrCP2 (SCF-

bTrCP2), these chains did not activate the RIG-I pathway. Simi-

larly, linear polyUb chains generated by Ubc5c and an E3

complex consisting of HOIP-1 and HOIL-1L (termed LUBAC)

did not lead to significant IRF3 dimerization. To further test if

ubiquitin chains of other linkages could support IRF3 dimeriza-

tion, we used a panel of ubiquitin mutants harboring a single

lysine (Figure 3E). 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 acti-

vation were those containing a lysine at position 63 (K63 only

and His6-K63 only). Collectively, these results clearly demon-

strate 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 (Extended Results and

Figure S3).

Short, Unanchored, K63-Linked Ubiquitin ChainsActivate RIG-ITo 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-Ub4;

Figure 4A). 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 and 3).

We then tested a panel of ubiquitin chains of different lengths

and linkages for their ability to activate RIG-I(N) (Figure 4B). Inter-

estingly, K63-Ub chains containing more than two ubiquitin

moieties potently activated RIG-I(N) (Figure 4B, 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) (Figures

4C and 4E). Similar titration experiments were also carried out

using full-length RIG-I in the presence of 50-pppRNA (Figures

4D and 4F). 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 z 680 nM), whereas K63-Ub2 was nearly inactive even

at the highest concentration (1 mM). 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.

The Tandem CARDs of RIG-I Bind to K63-UbiquitinChainsThe 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 (Figure 5A). Both CARDs 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

(Figures 5B and 5C; see also Figures S5A and 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 (Figure 5C, lane 10), suggesting that the signaling

defect of this mutant may be due to its failure to bind

K63-ubiquitin chains.

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�200 kDa (Figure 5D). When

each protein alone was fractionated on the same column,

K63-Ub4 and RIG-I(N) eluted at positions corresponding to

�25 and �50 kDa, respectively. These results suggest that the

binding of K63-Ub4 to RIG-I(N) promotes the formation of an

oligomeric complex (e.g., a tetramer).

Full-length RIG-I Binds to K63-Ubiquitin Chainsin a Manner that Depends on RNA and ATPAs full-length RIG-I is regulated by RNA binding and ATP hydro-

lysis, 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 coprecipitation of K63-Ub4 and long

K63-polyUb chains in the presence of 50-pppRNA and ATP

(Figure 5E, lanes 2 and 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 (Figure 5F). To

determine if RIG-I binds to RNA and K63 polyUb in a sequential

manner, we incubated RIG-I with 50-pppRNA or K63 polyUb in

reciprocal orders (Figure 5G). If RIG-I was preincubated with

50-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 prein-

cubated with K63 polyUb first, then immunoprecipitated to

remove unbound polyUb, its subsequent incubation with

50-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 Necessaryfor RIG-I ActivationPrevious studies have shown that RIG-I is ubiquitinated at K172

and that a mutation of this residue (K172R) impairs its ability to

induce IFN-b (Gack et al., 2007). Consistent with an important

role of K172 in RIG-I activation, we found that GST-RIG-I(N) con-

taining the K172R mutation failed to activate IRF3 in the in vitro

reconstitution assay (Figure 6A, 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 (Figure 6B). These results show that

the IRF3-activating function of RIG-I correlates well with its

ubiquitin-binding activity.

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-IFN-b-luciferase reporter cells

(Figure 6C), then pulled down these proteins with glutathione


Figure 4. Short, Unanchored K63-Ubiquitin Chains Potently Activate RIG-I(A) K63-Ub4 activates RIG-I(N). GST-RIG-I(N) (0.2 mM) was incubated with or without K63-Ub4 (0.3 mM), followed by IRF3 dimerization assay as outlined.

(B) K63, but not K48, ubiquitin chains activate RIG-I(N). Ubiquitin chains of defined lengths and linkages were incubated with GST-RIG-I(N), then IRF3 dimerization

assay was carried out as in (A). The quality of the ubiquitin chains was evaluated by immunoblotting (lower panel) or silver staining (see Figure S4).

(C) K63-ubiquitin chains potently activate RIG-I(N) in a chain length- and linkage-dependent manner. Different concentrations of Ub chains (0.01–1 mM) or ubiq-

uitin (0.1–10 mM) were tested for RIG-I(N) activation using the IRF3 dimerization assay.


Sepharose (Figure 6D). To distinguish ubiquitin chains covalently

conjugated to RIG-I from those noncovalently associated with

RIG-I, the Sepharose beads were washed with either phos-

phate-buffered saline (PBS) or a more stringent buffer containing

0.1% SDS and 1% deoxycholate (RIPA; Figure 6D). Immunoblot-

ting with a GST antibody showed that whereas 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 (Figure 6D, lower panel). Immunoblotting of the

pull-down proteins with a ubiquitin antibody showed an inter-

esting difference depending on the wash buffers (upper panel).

Whereas 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 IFN-b

(Figure 6C), 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 IFN-b.

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 (Figure 6E) (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 unan-

chored 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 ubiq-

uitin system, the same protein expressed in HEK293T cells was

able to activate IRF3 without preincubation with ubiquitin chains

(Figure 6E, bottom panel), probably because RIG-I(N) isolated

from human cells is already bound to endogenous polyUb chains

(e.g., lane 9 in Figure 6D). Interestingly, although vOTU removed

most of the ubiquitin chains from GST-RIG-I(N) (Figure 6E, lanes

6 and 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 CARDs may be dispensable for its

activity.

Unanchored K63-Polyubiquitin Chains Isolatedfrom Human Cells Are Potent Activators of RIG-IIn Figure 6E, 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 Extended 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

(D) Similar to (C), except that full-length His6-RIG-I and 50-pppRNA were used in

(E and F) IRF3 dimers in (C) and (D), respectively, were quantified using ImageQ

ubiquitin chains from HEK293T cells by employing two strategies

(Figure 7A). 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 (Figure 7B). 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�C–80�C

was sufficient to inactivate the RIG-I protein (lane 1, 3, and 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, and 6). When the GST-RIG-I(N) protein expressed

in HEK293T cells was heated at 70�C, it was still capable of acti-

vating 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 and 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 and 12).

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 endog-

enous 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 b-mercaptoethanol, indi-

cating that they contained unanchored C termini (Figure S7A).

Second, we incubated the endogenous ubiquitin chains with

IsoT and then measured their activity in the IRF3 dimerization

assay (Figure 7C). 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 preincubation 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 (Figure 7C, right panel).

Several bands in the range of 40–75 kDa, most prominently the

lieu of RIG-I(N) in the reactions.

uant, then plotted against the concentration of Ub or Ub chains.


Figure 5. RIG-I CARDs Bind K63-Ubiquitin Chains, and This Binding in Full-Length RIG-I Is Regulated by RNA and ATP

(A) RIG-I(N) binds specifically to K63-ubiquitin chains. GST or GST-RIG-I(N) was incubated with Ub3 containing K63, K48, or linear linkage, then pulled down with

glutathione Sepharose, followed by immunoblotting. Input represents 10% of Ub3 used in the pull-down experiments.

(B) Diagram of RIG-I N terminus containing the tandem CARDs and various deletion and point mutants. The table on the right summarizes the results in (C).

(C) Both CARDsof RIG-I are required for polyUb binding and IRF3 activation. GST-RIG-I(N) and various mutants were incubated with K63-Ub4.One microliter aliquot

of eachmixturewas used for IRF3 dimerization assay (upper panel), and the remainder was pulled down withglutathione Sepharose followed by immunoblottingwith

a Ub antibody (middle panel). The GST-RIG-I(N) and the mutant proteins (2 mg each) were analyzed by Coomassie blue staining (lower panel). See also Figure S5.

(D) RIG-I(N) and K63-Ub4 form an active high-molecular-weight complex. RIG-I(N) was incubated with K63-Ub4 and then the mixture was fractionated on Super-

dex-200. Aliquots of the fractions were assayed for their ability to stimulate IRF3 dimerization, whereas other aliquots were subjected to immunoblotting with

antibodies against RIG-I and ubiquitin. K63-Ub4 or RIG-I(N) alone was also analyzed by gel filtration on the same column (lower two panels).


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 treat-

ment, suggesting that they represent ubiquitin chains conju-

gated to some protein targets. Because the IsoT treatment

completely abolished the activity of the heat supernatant to

promote IRF3 dimerization (Figure 7C, 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 (Figure 7D, left panel). CYLD

also destroyed the ubiquitin chains with molecular masses in

the range of 40–75 kDa, which could be detected with an anti-

body specific for the K63-ubiquitin linkage (Figure 7D, right

panel).

TRIM25 and CYLD have previously been shown to be impor-

tant 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 Figure 7A. Immunoblotting experiments

showed that depletion of TRIM25 diminished, whereas depletion

of CYLD enhanced, the production of endogenous polyUb

chains, which in turn activated IRF3 (Figure 7E).

To evaluate the potency of endogenous polyUb chains in acti-

vating RIG-I, we carried out titration experiments using different

amounts of heat-resistant supernatants isolated from HEK293T

cells (Figure 7F). The amount of endogenous K63-Ub6 was

estimated by comparing to synthetic K63-Ub6 using semiquan-

titative immunoblotting (Figure S7B). Because K63-Ub6 is the

dominant species of endogenous polyUb chains, its concentra-

tion 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 (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 dimin-

ished by IsoT treatment (Figure S7D), indicating that the super-

(E) Full-length RIG-I binds to ubiquitin chains in a manner that depends on 50-pp

chains in the presence or absence of 50-pppRNA (135 nt), ATP, or EDTA. Follow

detected with an antibody against Ub or RIG-I. The asterisk indicates a nonspe

(F) Similar to (E), except that RIG-I ATPase mutants (K270A and D372N) we

nonspecific band.

(G) Sequential binding of RIG-I to RNA and polyUb leads to IRF3 activation. His8-

immunopurified using a Flag antibody. The purified RIG-I was incubated with K63-

natant 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 50-pppRNA

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 (1) the

unanchored K63-ubiquitin chains are present in human cells;

(2) they are bound and protected by RIG-I; and (2) 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 (Figure 7G).

DISCUSSION

In this report, we demonstrate that RNA with characteristics

of viral RNA (50-pppRNA and dsRNA) can activate the RIG-I

pathway in vitro in a reconstitution system with exquisite speci-

ficity and sensitivity. To our knowledge, this is the first in vitro

reconstitution of an immune signaling cascade from a microbial

ligand (viral RNA) to activation of a transcription factor (IRF3). Our

success in reconstituting the RIG-I pathway in vitro is beneficiary

of at least three previous discoveries that (1) RNAs containing

50-triphosphate are direct functional ligands of RIG-I (Hornung

et al., 2006; Pichlmair et al., 2006); (2) mitochondria containing

MAVS are essential for RIG-I signaling (Seth et al., 2005); and

(3) ubiquitination is important for RIG-I activation (Gack et al.,

2007). The in vitro reconstitution of the RIG-I pathway sets the

stage for delineating the biochemical mechanism of RIG-I activa-

tion and subsequent steps of downstream signaling. Testimonial

to the power of this in vitro system are the discoveries revealed in

this study that the tandem CARDs of RIG-I represent a new class

of ubiquitin-binding domain with specificity for K63-polyUb

chains and that unanchored K63-polyUb chains are potent

intracellular signaling molecules that activate the RIG-I pathway.

Specificity and Sensitivity of Viral RNA Detectionby RIG-IA major determinant that distinguishes viral RNA from the host

RNA in mammalian cells is the presence of 50-triphosphate in

viral RNA but not in host RNA (Fujita, 2009). 50-pppRNA is gener-

ated in the course of RNA replication by viral RNA polymerases.

However, RIG-I may have very limited access to viral 50-pppRNA

for the following reasons: (1) viral RNA replication normally

occurs within a membrane compartment (e.g., vesicles) that is

sequestered from the cytosolic immune sensors such as RIG-I;

(2) some viruses such as influenza replicate in the nucleus rather

pRNA and ATP. His8-RIG-I-Flag was incubated with K63-Ub4 or K63-polyUb

ing immunoprecipitation with a Flag antibody, the precipitated proteins were

cific band.

re also tested for binding to K63-polyUb chains. The asterisk indicates a

RIG-I-Flag was incubated with 50-pppRNA or K63-polyUb in the first step, then

polyUb or 50-pppRNA in the second step, followed by IRF3 dimerization assay.


Figure 6. Polyubiquitin Binding Is Required for RIG-I Activation

(A) Mutations at K172 and T55 impair RIG-I(N) activation in vitro. GST-RIG-I(N) and the indicated mutants were expressed and purified from E. coli, then incubated

with K63-Ub4, followed by IRF3 dimerization assay (upper panel). Aliquots of the reaction mixtures were analyzed by immunoblotting with a GST antibody (lower

panel). 6KR: GST-RIG-I(N) containing six K > R mutations, including K172R. K172-only: similar to 6KR except that K172 is not mutated.

(B) Mutations at K172 and T55 impair polyUb binding by RIG-I(N) in vitro. GST-RIG-I(N) and mutant proteins were incubated with K63-polyUb chains, then pulled

down with glutathione Sepharose and analyzed by immunoblotting.

(C) Mutations at K172 and T55 impair RIG-I(N)’s ability to induce IFN-b in cells. Different amounts (30 ng and 100 ng) of mammalian expression vectors for GST-

RIG-I(N) and mutants were transfected into HEK293-IFN-b-Luc cells, then luciferase activity was measured. Error bars represent the variation range of duplicate

experiments.

(D) GST-RIG-I(N) and mutant proteins as described in (C) were pulled down with glutathione Sepharose beads, washed with PBS or RIPA buffer, then analyzed by

immunoblotting.

(E) GST-RIG-I(N) was expressed in HEK293T cells, then purified using glutathione Sepharose. The purified protein was treated with isopeptidase T (IsoT) or viral

OTU (vOTU) or mock-treated, then immunoblotted with an antibody against ubiquitin or GST (upper panel; N.S indicates a nonspecific band). Aliquots of the

treated GST-RIG-I(N) were tested for their ability to promote IRF3 dimerization in the reconstitution assay (lower panel).

See also Figure S6.


than in the cytosol; (3) the majority of viral RNA is in complex with

viral capsid proteins to form ribonucleoprotein particles (RNPs),

and the 50 end of viral RNA can be blocked by viral proteins; (4)

like their mammalian counterparts, most viral mRNAs also

contain 50-cap or other 50-modifications. Thus, RIG-I must be

capable of detecting very tiny amounts of 50-pppRNA that may

be inadvertently or transiently exposed during the course of

viral replication and intracellular trafficking. By reconstituting

RNA-mediated activation of the RIG-I cascade in vitro, we

demonstrate that RIG-I can detect a few 50-pppRNA molecules

(<20 in a cell) in the presence of abundant cellular RNA and

trigger a signaling cascade leading to IRF3 activation. Thus,

our reconstitution system recapitulates the specificity and sensi-

tivity of viral RNA recognition by the RIG-I pathway in cells.

RIG-I CARDs Represent a New Type of Ubiquitin SensorCARDs are protein interaction domains that play key roles in

many cellular processes including inflammation and apoptosis

(Park et al., 2007). Unexpectedly, we found that the tandem

CARDs of RIG-I bind specifically to K63-linked polyUb chains.

We also found that the tandem CARDs of MDA5, but not the

CARDs of NOD2 or MAVS, bind to K63-polyUb chains (X.J.

and Z.C., unpublished data; Figure S5C). Thus, a subset of

CARDs appears to have the specialized ability to bind K63-poly-

Ub chains. Importantly, mutations that disrupt ubiquitin binding

of RIG-I also impair its ability to activate IRF3, indicating that

polyUb binding plays a key role in RIG-I activation. It will be of

significant interest to identify other CARDs capable of ubiquitin

binding and determine whether proteins harboring these CARDs

require ubiquitin binding for their functions.

Polyubiquitin Binding Is Required for RIG-I ActivationAn important role of RIG-I ubiquitination in its activation was

suggested based on the observations that K172R mutation of

RIG-I diminishes its ubiquitination and its ability to induce IFN-b

and that the K172-only mutant can induce IFN-b (Gack et al.,

2007). Further supporting this conclusion was the finding that

the T55I mutation, which was known to abrogate IFN-b induction,

also impairs RIG-I ubiquitination (Gack et al., 2008). However,

closer examination of these mutants reveals that K172R and

T55I mutations also impair the ability of RIG-I to bind K63-polyUb

chains (Figure 6B). Moreover, the K172-only mutation reinstalls

polyUb binding by RIG-I, but not ubiquitination of RIG-I

(Figure 6D), implying that polyUb binding but not ubiquitin

modification is required for RIG-I’s function. In support of this

notion, removal of ubiquitin chains from RIG-I with a DUB

(vOTU) had virtually no effect on RIG-I’s activity (Figure 6E).

Most importantly, we demonstrated that unanchored K63-poly-

Ub chains exist in human cells and that these endogenous

chains are potent ligands of RIG-I (Figure 7). Interestingly,

although the RIG-I complex isolated from HEK293T cells

contains both IsoT-sensitive unanchored ubiquitin chains and

IsoT-resistant ubiquitin conjugates, the IsoT treatment com-

pletely abolished the ability of these ubiquitin chains to activate

RIG-I (Figure 7C), strongly suggesting that unanchored polyUb

chains are responsible for RIG-I activation. Although we cannot

rule out the possibility that ubiquitination of some signaling

proteins may contribute to RIG-I activation, our data show that

ubiquitinated TRIM25 is incapable of activating RIG-I (Figure S3).

Regulation of RIG-I by Endogenous Polyubiquitin Chainsduring Viral InfectionWe estimated that approximately 10 ng of endogenous K63-Ub6

could be isolated from 20 million HEK293T cells using our

protocol (Figure S7B). This is equivalent to approximately 6000

molecules of K63-Ub6 per cell. These Ub chains activate RIG-I

with an EC50 of approximately 50 pM, which is equivalent to

�15 molecules per cell. This estimate, although imprecise,

suggests that there should be abundant endogenous Ub chains

to activate RIG-I(N), explaining why overexpression of RIG-I(N) is

sufficient to induce interferons in the absence of viral infection.

However, in normal cells containing endogenous full-length

RIG-I, which likely adopts an autoinhibited conformation, the

ubiquitin chains cannot gain access to the CARDs of RIG-I and

are rapidly disassembled by high levels of cellular DUB activity.

Viral infection or introduction of 50-pppRNA into the cytosol

presumably induces a conformational change of RIG-I that

exposes the CARDs, which then capture and stabilize unan-

chored K63-polyUb chains synthesized by TRIM25. We specu-

late that local and transient accumulation of these ubiquitin

chains in the vicinity of RNA-bound RIG-I allows the CARDs to

bind these chains, resulting in full activation of RIG-I. However,

as discussed above, only a very small fraction of RIG-I is acti-

vated by viral infection due to limited access to viral RNA in the

cytosol. Nevertheless, we obtained evidence that Sendai virus

infection led to association of IsoT-sensitive ubiquitin chains

with full-length RIG-I (Figures S7C and S7D). Given the high

potency of unanchored K63-ubiquitin chains in RIG-I activation,

accumulation of just a few molecules of these chains in a cell

is likely to activate endogenous RIG-I, provided that a few

molecules of viral RNA are also present.

Sequential Binding to RNA and UnanchoredK63-Polyubiquitin Chains Leads to FullActivation of RIG-IOur study reveals unanchored K63-polyUb chains as the second

ligand of RIG-I and demonstrates that full activation of RIG-I

requires an orderly binding of viral RNA followed by polyUb

chains. Based on available evidence, we propose a multistep

model of RIG-I activation (Figure 7G). After viral infection, RIG-I

binds to viral 50-pppRNA through its C-terminal RD domain

and may bind to the double-stranded segments of the RNA

through the central helicase domain (Fujita, 2009). The RNA

binding causes dimerization of RIG-I and activates its ATPase

activity (Cui et al., 2008). The energy derived from ATP hydrolysis

propels the translocation of RIG-I along the viral RNA (Myong

et al., 2009). The translocation of RIG-I on viral RNA may help

displace viral proteins from the RNA, facilitating further RIG-I

binding. The translocating RIG-I dimer likely has an ‘‘open’’

conformation that exposes the N-terminal CARDs, which form

a cluster consisting of four CARDs. This cluster of CARDs binds

to K63-polyUb chains. This binding may cause additional confor-

mational changes and/or oligomerization of RIG-I, forming a new

signaling platform to activate MAVS. The dependency of RIG-I


Figure 7. Endogenous Unanchored K63-Polyubiquitin Chains Activate the RIG-I Pathway

(A) A protocol for isolating functional endogenous polyUb chains in human cells.

(B) Endogenous polyUb chains bound to RIG-I can be released by heat treatment and remain functional. GST-RIG-I(N)-K172-only was expressed in HEK293T

cells and isolated as described in (A), then heated for 5 min at the indicated temperatures. After centrifugation, the supernatant containing heat-resistant ubiquitin

chains was incubated with GST-RIG-I(N) expressed and purified from E. coli (lanes 7–12). The activity of GST-RIG-I(N) was then measured by IRF3 dimerization

assay. As positive controls, K63-polyUb chains were incubated with GST-RIG-I(N)-K172-only, which was then pulled down and heated in parallel experiments

(lanes 1–6). Endo.polyUb: endogenous polyUb.

(C) Endogenous unanchored polyUb chains activate RIG-I(N). PolyUb chains associated with GST-RIG-I(N)-K172-only were captured and released at 75�C as in

(B). The heat-resistant supernatant was incubated with GST-RIG-I(N) followed by IsoT treatment (lane 9), or in reverse order (lane 8). As positive controls,


activation on binding to unanchored K63-ubiquitin chains may

provide another level of regulation of antiviral immune responses.

Unanchored K63-Polyubiquitin ChainsAre Intracellular Signaling MoleculesOur previous study has shown that unanchored polyUb chains

directly activate the protein kinases TAK1 and IKK by binding

to the essential regulatory subunits TAB2 and NEMO, respec-

tively (Xia et al., 2009). We now show that unanchored polyUb

chains bind and activate RIG-I, suggesting that these ubiquitin

chains may have more general functions in cell signaling.

However, unlike TAK1 and IKK activation, which requires very

long polyUb chains, RIG-I can be activated by short K63 chains

consisting of only 3 or 4 ubiquitin moieties. Indeed, K63-Ub4 and

endogenous Ub chains are very potent activators of RIG-I(N)

(Figure 4E and Figure 7F). The clusters of multiple CARDs at

the N termini of RIG-I may bind to K63-ubiquitin chains with

strong affinity and high selectivity. This is in contrast to most

ubiquitin-binding domains, which bind to ubiquitin chains with

weak affinity and low selectivity (Hurley et al., 2006). Our finding

that short ubiquitin chains associate with and potently activate

RIG-I in cells strongly suggests that these are endogenous

ligands of RIG-I. These findings present an unprecedented

example of a biological function of short unanchored ubiquitin

chains and deviate from the paradigm that ubiquitin functions

through covalent conjugation of another protein. It will be of

enormous interest to investigate whether unanchored polyUb

chains regulate other physiological functions besides the RIG-I

and NF-kB pathways.

EXPERIMENTAL PROCEDURES

Biochemical Assay for RIG-I Activation In Vitro

For the standard assay with full-length RIG-I, each ml mixture contained 10 ng

His6-E1, 15 ng Ubc5c, 50 ng His8-TRIM25, 1 mg ubiquitin, indicated amounts of

RNA (200 ng poly(I:C), 30 ng 50-pppRNA [79nt] or 50 ng 50-pppRNA [135nt]),

and 50 to 100 ng His8-RIG-I-Flag in Buffer A (20 mM Tris-HCl [pH 7.5], 2 mM

ATP, 5 mM MgCl2, and 0.5 mM DTT). After incubation at 30�C for 1 hr, 1 ml

of reaction mixture was directly used in IRF3 activation assay as described

in the Extended Experimental Procedures (see also Zeng et al., 2009). For

RIG-I activation assays containing mutant ubiquitin, 1 ml of reaction mixture

was mixed with 10 mg of mitochondrial fraction (P5) in 10 ml Buffer B (20 mM

HEPES-KOH [pH 7.0], 2 mM ATP, 5 mM MgCl2, and 0.25 M D-mannitol) at

30�C for 30 min. P5 was then pelleted at 10,000 g for 10 min and washed twice

with Buffer C (20 mM HEPES-KOH at pH 7.4, 0.5 mM EGTA, 0.25 M

D-mannitol, and EDTA-free protease inhibitor cocktail) before it was used for

the IRF3 activation assay.

unanchored K63-polyUb chains were incubated with GST-RIG-I(N) and IsoT in se

endogenous polyUb from HEK293T cells was incubated with or without IsoT, the

�40 kDa band that is likely K63-Ub6 (see Figure S7B).

(D) Similar to (C), except that the supernatant containing endogenous polyUb cha

antibody or another antibody specific for the K63 linkage of ubiquitin chains.

(E) siRNA oligos targeting GFP (control), TRIM25, or CYLD were transfected into H

encoding GST-RIG-I(N)-K172-only. Endogenous polyUb chains associated with

dimerization assay and visualized by immunoblotting with a ubiquitin antibody. T

(F) Potent activation of RIG-I by endogenous polyUb chains. Different amounts of h

GST-RIG-I(N) to measure IRF3 dimerization. The concentration of the ubiquitin cha

represent the variation range of duplicate experiments.

(G) A proposed mechanism of RIG-I activation by RNA and polyUb (see Results

For the standard assay with RIG-I (N), each ml mixture contained 100 ng

GST-RIG-I(N) and 50 to 100 ng ubiquitin chains in a buffer containing 20

mM HEPES-KOH (pH 7.0) and 10% (v/v) glycerol. After incubation at 4�C

for 10 min or up to 1 hr, the mixture was directly used in the IRF3 activation

assay.

Ubiquitin-Binding Assay

For ubiquitin binding by full-length RIG-I, His8-RIG-I-Flag (1 mg) or the ATPase

mutant (K270A or D372N) was incubated with K63-Ub4 or K63-polyUb (1 mg),

and 50-pppRNA (0.3 mg for 135nt RNA) in 10 ml Buffer A at 4�C for 1 hr. For

reactions lacking ATP, EDTA (10 mM) was added. The mixture was then diluted

5-fold in Buffer D (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.5% [w/v] NP-40,

and 10% [v/v] glycerol) and immunoprecipitated with anti-Flag or anti-RIG-I

antibody. The bound proteins were eluted with Flag peptide or SDS sample

buffer and subjected to immunoblotting analysis with antibodies specific for

ubiquitin or RIG-I.

For ubiquitin binding by RIG-I(N), 2 mg of wild-type or mutant GST-RIG-I(N)

was incubated with 1 mg ubiquitin chains in 10 ml buffer containing 20 mM

HEPES-KOH (pH 7.0) and 10% (v/v) glycerol at 4�C for 1 hr. The mixture

was then diluted 5-fold in Buffer D before the GST proteins were pulled

down with glutathione Sepharose. The bound proteins were analyzed by

immunoblotting.

To analyze RIG-I(N):K63-Ub4 complex, GST-RIG-I(N) was cleaved with

Tev protease and then RIG-I(N) was purified on Q-Sepharose and Super-

dex-200 columns. Five micrograms each of purified RIG-I(N) and K63-Ub4

were incubated at 4�C for 60 min, and then the mixture was separated on

Superdex-200 PC (3.2/30) using the SMART purification system. The elution

buffer contained 20 mM Tris-HCl (pH7.5), 0.1 mM EGTA, 0.5 mM DTT, and

0.02% CHAPS. Aliquots of the fractions were analyzed in the in vitro IRF3

dimerization assay and also by immunoblotting using antibodies against

RIG-I or ubiquitin.

Isolation and Functional Analysis of Endogenous

Polyubiquitin Chains

HEK293T cells were transfected in 10 cm dishes with 5 mg of pEF-GST-RIG-

I(N)-K172-only. The cells were lysed 48 hr later in Buffer E (20 mM Tris-HCl

at pH 7.5, 150 mM NaCl, 10% [v/v] glycerol, 1% [w/v] Triton X-100, 20 mM

b-glycerol phosphate, 1 mM Na3VO4, 0.1 mM NaF, 0.1 mM phenylmethanesul-

fonyl fluoride, and EDTA-free protease inhibitor cocktail) supplemented with

10 mM NEM. After centrifugation at 20,000 g for 15 min, 5 mM DTT was added

to the supernatant to quench NEM, then the GST proteins were isolated with

glutathione Sepharose. The proteins on the beads were incubated in 30 ml

Buffer F (20 mM HEPES-KOH at pH 7.0, 10% [v/v] glycerol, and 0.02% [w/v]

CHAPS) at 70�C, 75�C, or 80�C for 5 min. Following a brief centrifugation,

the supernatant containing the dissociated polyUb chains was incubated

with 100 ng of bacterially expressed GST-RIG-I(N) at 4�C for 1 hr, followed

by IRF3 dimerization assay. For deubiquitination enzyme treatment, the

heated supernatant was incubated with 10 ng/ml IsoT or CYLD at 30�C for

1 hr. The concentration of endogenous polyUb chains was estimated by

semiquantitative immunoblotting using synthetic K63-Ub6 (Boston Biochem)

as the standard (Figure S7B).

quential orders as indicated. In the right panel, the heat supernatant containing

n analyzed by immunoblotting with a ubiquitin antibody. The arrow denotes an

ins was treated with CYLD. The ubiquitin chains were detected with a ubiquitin

EK293T cells, which were subsequently transfected with an expression vector

the GST-RIG-I(N) protein were isolated as described in (A), then tested in IRF3

he efficiency of RNAi was also confirmed by immunoblotting.

eat-resistant supernatant containing endogenous polyUb were incubated with

ins was estimated by semiquantitative immunoblotting (Figure S7B). Error bars

and Discussion).


SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Results, Extended Experimental

Procedures, and seven figures and can be found with this article online at

doi:10.1016/j.cell.2010.03.029.

ACKNOWLEDGMENTS

We thank Francesco Melandri and Dr. Kal Lapan (Boston Biochem) for gener-

ously providing some of the ubiquitin chains and Dr. Jae Jung (University of

Southern California) for the mammalian expression plasmids of GST-RIG-

I(N), K172R, K172-only, and 6KR mutants. We also thank Brian Skaug for

reading the manuscript. This work was supported by grants from NIH

(RO1-AI09919 and RO1-GM63692) and the Welch Foundation (I-1389).

Z.J.C. is an Investigator of Howard Hughes Medical Institute.

Received: December 20, 2009

Revised: February 22, 2010

Accepted: March 23, 2010

Published: April 15, 2010

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http://dx.doi.org/doi:10.1016/j.cell.2010.03.029

NLRC5 Negatively Regulates the NF-kBand Type I Interferon Signaling PathwaysJun Cui,1,2,5 Liang Zhu,1,3,5 Xiaojun Xia,1,5 Helen Y. Wang,1 Xavier Legras,1 Jun Hong,1 Jiabing Ji,1 Pingping Shen,2

Shu Zheng,3 Zhijian J. Chen,4 and Rong-Fu Wang1,*1Center for Cell and Gene Therapy, and Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA2The College of Life Science, Nanjing University, Nanjing, People’s Republic of China3Cancer Institute, The Second Affiliated Hospital, Zhejiang University Medical School, Hangzhou, People’s Republic of China4Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas,

Texas 75390, USA5These authors contributed equally to this work

*Correspondence: [email protected] 10.1016/j.cell.2010.03.040

SUMMARY

Stringent control of the NF-kB and type I inter-feron signaling pathways is critical to effective hostimmune responses, yet the molecular mechanismsthat negatively regulate these pathways are poorlyunderstood. Here, we show that NLRC5, a memberof the highly conserved NOD-like protein family,can inhibit the IKK complex and RIG-I/MDA5 func-tion. NLRC5 inhibited NF-kB-dependent responsesby interacting with IKKa and IKKb and blocking theirphosphorylation. It also interacted with RIG-I andMDA5, but not with MAVS, to inhibit RLR-mediatedtype I interferon responses. Consistent with theseobservations, NLRC5-specific siRNA knockdownnot only enhanced the activation of NF-kB and itsresponsive genes, TNF-a and IL-6, but also pro-moted type I interferon signaling and antiviral immu-nity. Our findings identify NLRC5 as a negative regu-lator that blocks two central components of theNF-kB and type I interferon signaling pathways andsuggest an important role for NLRC5 in homeostaticcontrol of innate immunity.

INTRODUCTION

The innate immune response, elicited through the detection of

pathogen-associated molecular patterns (PAMPs), provides the

first line of defense against invading microorganisms. PAMP

recognition depends on several classes of pattern recognition

receptors (PRRs), including Toll-like receptors (TLRs), NOD-like

receptors (NLRs), and RIG-I-like receptors (RLRs) (Akira et al.,

2006; Honda and Taniguchi, 2006; Inohara et al., 2005; Medzhi-

tov, 2007; Meylan et al., 2006; Ting et al., 2006). Activation of

most TLRs leads to the recruitment of a common adaptor,

MyD88, and in turn to a series of downstream signaling events

that culminate in NF-kB activation (Akira et al., 2006; Chen,

2005; Hayden and Ghosh, 2008). By contrast, activation of

RLRs (RIG-I and MDA5) by double- and single-stranded RNAs

or certain viruses (Hornung et al., 2006; Kato et al., 2006; Pichl-

mair et al., 2006) results in recruitment of the MAVS protein (mito-

chondrial antiviral signaling; also called VISA, IPS-1 and Cardif),

which further activates the downstream signaling molecules

TBK1/IKKi and IRF3 for type I interferon responses, as well as

IKK molecules for NF-kB activation (Meylan et al., 2006). Besides

their roles in innate immunity and inflammation, TLR-mediated

signaling pathways have been shown to play an important role

in the control of regulatory T cell function (Liu et al., 2006;

Peng et al., 2005, 2007; Sutmuller et al., 2006).

Because uncontrolled immune responses can be harmful,

even fatal, to the host (Liew et al., 2005), NF-kB activation and

type I interferon signaling must be tightly regulated to maintain

immune balance in the organism. Despite the importance of

the IKK complex as a central transducer of signaling from various

stimuli, leading to the activation of the NF-kB pathway, and of

RLRs as critical receptors in type I interferon signaling (Chen,

2005; Honda and Taniguchi, 2006), the molecular mechanisms

responsible for IKK activation and RLR-mediated signaling

remain poorly understood.

NLRs represent a large family of intracellular PRRs that are

characterized by a conserved nucleotide-binding and oligomer-

ization domain (NOD) and a leucine-rich repeat (LRR) region, and

are involved in the activation of diverse signaling pathways (Akira

et al., 2006; Inohara et al., 2005; Meylan et al., 2006). Several

NLRs, such as NOD1, NOD2 and NALP3, have been extensively

studied and shown to activate signaling pathways once they

encounter relevant PAMPs (Akira et al., 2006; Chen, 2005;

Inohara et al., 2005; Meylan et al., 2006). NALP3 inflammasome,

for example, functions as a crucial component in the adjuvant

effect of aluminum and asbestos (Dostert et al., 2008; Eisenbarth

et al., 2008). More recently, NLRX1 was demonstrated to func-

tion as a mitochondrial protein that interacts with the mitochon-

drial adaptor MAVS to inhibit the RIG-I-mediated signaling

pathway and triggers the generation of reactive oxygen species

as well (Moore et al., 2008; Tattoli et al., 2008). These studies

suggest that understanding the function and mechanisms of

these innate immune receptors or regulators may aid in devel-

oping more effective strategies for the immunological treatment


mailto:[email protected]

Figure 1. Domain Organization, Expression,

and Intracellular Localization of Human and

Mouse NLRC5

(A) Domain organization of human and mouse

NLRC5 proteins. CARD, caspase recruitment

domain; NOD, nucleotide binding domain; LRR,

leucine rich repeats.

(B) Western blot analysis of HA-tagged NLRC5

and mNLRC5 protein expression in 293T cells.

(C) Human and mouse NLRC5 mRNAs were deter-

mined in different tissues determined by real-time

PCR analysis.

(D) Expression of mNLRC5 mRNA in RAW264.7

cells was determined by real-time PCR analysis

after LPS, intracellular poly (I:C) stimulation or

VSV-eGFP infection.

(E) Immunoblot analysis of mNLRC5 protein

expression after LPS stimulation or VSV-eGFP

infection (left panel). The relative protein levels of

mNLRC5 were quantified by the band density

scanning of western blot images and plotted

against time (hr) after LPS treatment or VSV infec-

tion (right panel).

(F) Both wild-type and MyD88-deficient macro-

phages were treated with LPS, and mNLRC

mRNA expression was determined by real-time

PCR analysis.

See also Figure S1.

of inflammation-associated diseases (Karin et al., 2006; Wang

et al., 2008).

Given that the NLR protein family is involved in many biological

processes and functions as proinflammatory receptors as well

as negative regulators, we hypothesized that some NLR mem-

bers may play a critical regulatory role in the control of NF-kB

and type I interferon signaling. Here we report the identification

of NLRC5 as a potent negative regulator of NF-kB and IRF3

activation. It strongly inhibits NF-kB-dependent responses by

interacting with IKKa and IKKb and blocking their phosphoryla-

tion. It also interacts with RIG-I and MDA5, but not with MAVS,

to potently inhibit RLR-mediated type I interferon responses.

As a key negative regulator of NF-kB and type I interferon sig-

naling, NLRC5 may serve as a useful target for manipulating

immune responses against infectious or inflammation-associ-

ated diseases, including cancer.

RESULTS

Molecular Cloning and Characterization of NLRC5As a member of the NLR protein family, NLRC5 contains a

CARD-like domain, a central NOD domain and a large LRR


E

o

u

e

th

d

e

a

H

c

c

ti

o

re

N

R

g

p

F

m

in

region (Figure 1A), but its biological func-

tion remains unknown (Chen et al., 2009;

Dowds et al., 2003). To determine the

function of NLRC5, we cloned full-length

human and murine NLRC5 complemen-

tary DNAs (cDNAs) by rapid amplification

of cDNA ends (RACE) (Figure 1A). There

was a 64% amino acid sequence identity

between the human and murine proteins.

xpression of both HA-tagged NLRC5 and mNLRC5 was dem-

nstrated by western blotting with an anti-HA antibody (Fig-

re 1B). Both human and murine NLRC5 mRNAs were strongly

xpressed in spleen, thymus, and lung (Figure 1C), suggesting

at this molecule is biologically conserved in these tissues.

To further demonstrate the protein expression of NLRC5 in

ifferent cell types, we generated polyclonal antibodies against

ndogenous NLRC5 and mNLRC5 (Figures S1A–S1E avail-

ble online), which readily detected these proteins in human

EK293T (293T) cells, human THP1 cells or mouse RAW264.7

ells (Figure S1F). To determine whether NLRC5 expression

ould be regulated in response to TLR stimulation or virus infec-

on, we treated RAW264.7 cells with LPS, intracellular poly(I:C),

r infection with vesticular stomatitis virus-enhanced green fluo-

scent protein (VSV-eGFP), which are known to activate the

F-kB or type I IFN pathway (Yoneyama and Fujita, 2009).

eal-time PCR and western blot analyses revealed strong upre-

ulation of mNLRC5 at both the mRNA and protein levels, which

eaked at 6 hr after LPS treatment (Figures 1D and 1E and

igure S1G). In contrast, we observed only a weak increase of

NLRC5 mRNA and little or no change in mNLRC5 protein after

tracellular poly(I:C) treatment or VSV infection (Figures 1D and

Figure 2. NLRC5 Inhibits NF-kB Activation

Induced by IL-1b, LPS, TNF-a, and Their

Downstream Signaling Molecules

(A) 293T cells were transfected with an NF-kB-luc

reporter plasmid and TLR4 plasmid (for LPS treat-

ment), together with an empty vector or NLRC5

construct, and analyzed for NF-kB-dependent

luciferase activity (fold induction) after treatment

with IL-1b, LPS, or TNF-a.

(B) 293T cells were transfected with MyD88,

TRAF6, IKKa, IKKb, or p65, along with NF-kB-luc.

(C) 293T cells were transfected with NOD1 or

NOD2, along with NF-kB-luc.

(D) Detection of endogenous NF-kB DNA binding

activity in a gel-mobility shift assay. Oct-1/DNA-

binding complexes served as a loading control

for nuclear extracts.

(E) Human THP-1 cells and murine embryonic

fibroblasts (MEF) were transfected with the

NF-kB-luc reporter plasmid, together with (or

without) NLRC5 or mNLRC5 plasmid, and then

analyzed for NF-kB-dependent luciferase activity

after LPS treatment (***p < 0.001).

See also Figure S2.

1E), consistent with a previous study showing that VSV infection

is a weak NF-kB inducer (Ishikawa and Barber, 2008). To further

explore the mechanisms by which NLRC5 is regulated, we per-

formed similar experiments with wild-type and MyD88 knockout

(KO) macrophages and found that upregulation of mNLRC5 was

completely abolished when MyD88 KO macrophages were

stimulated with LPS (Figure 1F), indicating that the expression

of mNLRC5 itself is controlled by the NF-kB signaling pathway.

To determine the cellular localization of NLRC5, we trans-

fected 293T cells with NLRC5-GFP fusion DNA and found that

NLRC5-GFP was present in the cytoplasm, but not in the nucleus

or mitochondria (Figures S1H–S1I), suggesting that NLRC5 is

a cytosolic protein. No change in the cellular localization of

NLRC5 was observed after LPS treatment (Figures S1I–S1K).

NLRC5 Is a Potent Negative Regulator of NF-kBActivation Induced by TLR LigandsTo determine whether NLRC5 is involved in TLR- and/or

cytokine-mediated NF-kB activation, we transfected 293T or

293T/TLR4 cells with NF-kB-luc reporter DNA, with or without

Cell 141, 483–4

the NLRC5 plasmid, and then treated

them with interleukin (IL)-1b, TNF-a or

LPS. Results in Figure 2A show that

NLRC5 potently inhibited NF-kB activa-

tion induced by IL-1b, TNF-a or LPS.

We next sought to identify potential sig-

naling molecules that activated the NF-

kB-luc reporter. Expression of MyD88,

TRAF6, IKKa, or IKKb strongly induced

NF-kB-luc activity, but such activity was

inhibited when NLRC5 was cotransfected

at increasing concentrations (Figure 2B).

In contrast, NLRC5 did not inhibit p65-

mediated NF-kB-luc activity, suggesting

that it may interact with IKK signaling molecules upstream of

p65 to block NF-kB activation (Figure 2B). The control plasmids

NOD1 and NOD2 enhanced rather than inhibited NF-kB activity

(Figure 2C), consistent with previous studies (Inohara et al.,

1999; Ogura et al., 2001).

Since LPS can also activate the type I IFN pathway through an

adaptor TRIF molecule, we asked whether NLRC5 can inhibit

LPS-induced IFN-b-luc activity (which requires cooperation

between IRF3 and NF-kB activation) or interferon-stimulated

response element (ISRE)-luc activity (which requires IRF3 activa-

tion only) (Zhong et al., 2008). Functional assays showed that

NLRC5 inhibited LPS- or TRIF-induced NF-kB-luc and IFN-b-

luc activities, but had no effects on ISRE-luc activity (Figures

S2A and S2B). Further experiments demonstrated that NLRC5

strongly inhibited R848-induced NF-kB-luc activity and weakly

inhibited IFN-b-luc activity, but did not inhibit ISRE-luc activity

(Figure S2C). Thus, NLRC5 strongly inhibits NF-kB activation

induced by different TLR ligands and weakly blocks IFN-b activa-

tion mainly due to its requirement for NF-kB activity, but has no

apparent effect on ISRE activity.

96, April 30, 2010 ª2010 Elsevier Inc. 485

To determine whether the observed NLRC5-mediated inhibi-

tion of NF-kB-luc activity is associated with endogenous NF-kB

activity, we performed experiments with a gel-mobility shift

assay. IKKb expression allowed endogenous NF-kB to bind to

the biotin-HRP-labeled DNA with NF-kB binding sites, but this

activity was completely inhibited when NLRC5 was cotrans-

fected. However, NLRC5 failed to inhibit p65-mediated NF-kB

activation (Figure 2D), consistent with results obtained with the

NF-kB-luc reporter assay. To substantiate these findings, we

found that like its human homolog, mNLRC5 strongly inhibited

NF-kB activation by MyD88, TRAF6, IKKa and IKKb, but not

by p65 (Figure S2D). Moreover, NLRC5- or mNLRC5-mediated

NF-kB inhibition in 293T cells could be extended to THP-1 cells

and murine embryonic fibroblasts (MEFs) (Figure 2E). Taken

together, these results suggest that NLRC5 functions as a nega-

tive regulator of NF-kB activation induced by TNF-a, IL-1b, LPS

or by their downstream signaling molecules, and that its biolog-

ical function is conserved between humans and mice as well as

among multiple cell types.

NLRC5 Interacts with IKKa/IKKb in Unstimulatedand Stimulated CellsResults presented in Figures 2B and 2D suggest that NLRC5

may directly interact with IKKa, IKKb or NEMO to inhibit NF-kB

activation. To test this prediction, we transfected 293T cells

with HA-tagged NLRC5 together with Flag-tagged IKKa, Flag-

tagged IKKb or Flag-tagged NEMO expression plasmids. Coim-

munoprecipitation and western blot analyses revealed that

NLRC5 interacted with IKKa and IKKb subunits, but not with

NEMO, although the corresponding proteins were readily

detected in whole-cell lysates (Figure 3A). Notably, NLRC5 did

not interact with either TAK1 or MEKK3 upstream of IKK

(Figure S3A). To further test whether NLRC5 can interact with

endogenous IKKa/b, we immunoprecipitated 293T cell lysates

with an isotype IgG or anti-NLRC5 antibody, followed by western

blot analysis with the anti-IKKa/b, anti-NEMO or anti-NLRC5

antibody. As shown in Figure 3B, the IKKa/b, but not NEMO,

could be detected in the anti-NLRC5 or anti-mNLRC5-immuno-

precipitants. Such an interaction between NLRC5 and IKKa/b

was also observed when anti-IKKa/b and anti-NEMO antibodies

were used for immunoprecipitation (Figure S3B). These results

suggest that NLRC5 can interact with IKKa/b, but not with

NEMO, under physiological conditions.

Since IKKa/b generally forms a complex with NEMO, a key

question is whether there are two distinct complexes in unstimu-

lated cells: IKKa/b/NEMO and IKKa/b/NLRC5. To address this

issue, we used different antibodies (anti-IKKa, anti-Flag and

anti-NEMO) to immunoprecipitate the IKK complexes containing

either NLRC5 or NEMO, and then determined the components of

their complexes. We found NEMO and NLRC5 in the anti-IKKa

immunoprecipitants, NLRC5 and IKKa/b (but not NEMO) in the

anti-Flag-NLRC5 immunoprecipitants, and IKKa/b and NEMO

(but not NLRC5) in the anti-NEMO immunoprecipitants (Fig-

ure S3C), suggesting that the IKKa/b/NEMO and IKKa/b/

NLRC5 complexes coexist in unstimulated cells. To definitively

demonstrate the presence of two distinct complexes, we frac-

tionated cell extracts of RAW264.7 on a size-exclusion column

(HiPrep 16/60 Sephacryl S-300 HR). Immunoblotting of fractions


collected from chromatography showed that IKKa/b mainly coe-

luted with NEMO in fractions 22–31 (molecular mass about 700

kDa) (Figure 3C), consistent with a previous report (Rothwarf

et al., 1998). In contrast, NLRC5 mainly eluted in fractions 15-18

(molecular mass about 1100 kDa), which also contained IKKa/

b but no NEMO (Figure 3C), suggesting that NLRC5 forms

a larger complex with IKKa/b than previously reported for the

IKKa/b/NEMO complex, thus confirming the coexistence of

IKKa/b/NLRC5 and IKKa/b/NEMO complexes in unstimulated

cells.

To determine the dynamics of NLRC5 interaction with IKKa/

b after stimulation, we treated RAW264.7 cells with LPS, col-

lected them at different time points and performed immunopre-

cipitation and western blot analyses. The interaction between

NLRC5 and IKKa/b was reduced at 30 min, but was restored

at 60 min after LPS stimulation (Figure S4A). This oscillating

pattern of interaction correlated inversely with IKK phosphoryla-

tion (p-IKK) during the first 60 min posttreatment and then dimin-

ished or even disappeared after 2 hr of treatment (Figures S4A–

S4C), suggesting that the negative regulatory activity of NLRC5

is signal-dependent, but is not affected exclusively by protein

concentration.

NLRC5 Competes with NEMO for IKKa/IKKb and InhibitsTheir Phosphorylation and Kinase ActivityWe next tested whether NLRC5 and NEMO compete each other

for binding to IKKa/b. 293T cells were transfected with a fixed

concentration of Flag-NLRC5 and HA-IKKb DNAs, together

with increasing concentrations of HA-NEMO DNA. Coimmuno-

precipitation and immunoblot revealed that the binding between

NLRC5 and IKKb was markedly decreased with increasing con-

centrations of NEMO (Figure 3D). Although the phosphoryla-

tion and kinase activity of IKKa/b could be detected in the

IKKa/b/NEMO complex after LPS stimulation, they decreased

with increasing amounts of transfected NLRC5. There were no

detectable phosphorylation and kinase activity of IKKa/b in

the IKKa/b/NLRC5 complex (Figure 3E). Importantly, increasing

amounts of NLRC5 had more pronouced effects on the phos-

phorylation and kinase activity of IKKa/b in the total and

NEMO-immunoprecipitated IKKa/b fraction than on its changes

at the protein level (Figure 3E). These results suggest that NLRC5

not only competes with NEMO for IKKa/b binding, but also

inhibits IKKa/b phosphorylation and its ability to phosphorylate

IkB or free IKKa/IKKb. To test this possibility, we performed

experiments with constitutively active IKKa (SS176/180/EE)

or IKKb (SS177/181/EE) mutants and found that NLRC5 inter-

acted with constitutively active IKKa (EE) and IKKb (EE) (Fig-

ure S4D). Kinase assays revealed that expression of NLRC5

inhibited the ability of IKKa (EE) and IKKb (EE) to phosphorylate

IkB (i.e., 32p-GST-IkBa), as well as their ability to autophosphor-

ylate IKK (i.e., 32p-IKK) (Figure S4E). These observations were

further supported by NF-kB-luc assays showing that NLRC5

can inhibit NF-kB activation by the constitutively active IKKa

(EE) and IKKb (EE) (Figure S4F). These results suggest that

NLRC5 can inhibit the ability of active IKKa/IKKb to phosphory-

late IkBa or free IKKa/IKKb.

To determine how NLRC5 inhibits the phosphorylation of

IKK, we sought to identify the domain of IKKb responsible

Figure 3. NLRC5 Interacts with IKKa and IKKb to Inhibit Their Phosphorylation

(A) 293T cells transfected with Flag-IKKa, Flag-IKKb, Flag-NEMO and HA-NLRC5. HA-tagged NLRC5 protein was immunoprecipitated with anti-HA beads, and

blotted with anti-Flag.

(B) 293T and RAW264.7 cell extracts were immunoprecipitated with IgG, anti-NLRC5 or anti-mNLRC5 antibody, respectively, and then analyzed together with

whole-cell extracts by western blot with an anti-IKKa/b, anti-NEMO, anti-NLRC5 or anti-mNLRC5 antibody.

(C) Cell extracts of RAW264.7 cells were fractionated on a size-exclusion column (HiPrep 16/60 Sephacryl S-300 HR). The collected factions with an equal volume

were used for western blot analysis with specific antibodies. The elution positions of calibration proteins with known molecular masses (kDa) were used to deter-

mine the size of complexes.

(D) 293T cells were transfected with the indicated doses of Flag-NLRC5, HA-IKKb and HA-NEMO. Whole-cell extracts were immunoprecipitated with anti-Flag

beads and blotted with anti-HA.

(E) 293T/TLR4 cells were transfected with empty vector or different doses of HA-NLRC5 (0, 50 or 200 ng) DNA and then treated with LPS. Cell extracts were

collected at 30 min poststimulation and prepared for immunoprecipitation with anti-HA and anti-NEMO, followed by immunoblot (IB) with indicated antibodies

or kinase assay (KA).

(F) The domain structure of IKKb. Numbers in parentheses indicate amino acid position in construct. LZ, leucine zipper; HLH, helix-loop-helix.

(G) 293T cells were transfected with HA-NLRC5 and Flag-IKKb or various Flag-IKKb mutants. Whole-cell extracts were immunoprecipitated with anti-Flag beads,

and blotted with anti-HA.

(H and I) 293T cells transfected with IKKa, IKKb, JNK1, JNK2, and p38 with or without HA-NLRC5 were used to analyze the phosphorylation of IKKa/b, JNK and p38.

(J) RAW264.7 cells were treated with LPS and collected at the indicated time points. Cell extracts were prepared for immunoprecipitation with anti-mNLRC5 or anti-

NEMO, followed by immunoblot (IB) to determine the IKK phosphorylation with anti-p-IKK or anti-IKKa/b antibody and kinase activity of IKK.

See also Figure S3 and Figure S4.


Figure 4. Interaction and Functional Anal-

ysis of NLRC5 Deletions in the Inhibition of

NF-kB Activation

(A) Four NLRC5 deletion constructs were

generated.

(B and C) 293T cells were transfected with NLRC5

and its mutations constructs, IKKb or kinase

domain of IKKb and analyzed by coimmunopreci-

pitation and western blot.

(D) 293T cells were transfected with NF-kB-luc

reporter, together with an empty vector, or with

full-length NLRC5 and its deletion constructs,

and analyzed for luciferase activity (fold induction).

(E) 293T cells were transfected with various

expression plasmids and the phosphorylation of

IKKa or IKKb was determined.

See also Figure S4G.

for interacting with NLRC5. Because NEMO is known to bind

to the C terminus of IKKb (May et al., 2000), we generated dele-

tion mutants encompassing the amino-terminal kinase domain

(KD), leucine zipper domain (LZ) and a C-terminal helix-loop-

helix (HLH) domain of IKKb, and tested them for their ability

to interact with NLRC5 in an immunoprecipitation assay (Fig-

ure 3F). Like the full-length IKKb, the IKKb-KD construct strongly

interacted with NLRC5, while neither the IKKb-LZ nor the

IKKb-HLH construct showed appreciable binding activity with

NLRC5 (Figure 3G), indicating that NLRC5 specifically binds to

the IKKb-KD domain. However, because of the large size of

the NLRC5 protein, we considered that its binding to IKKb

might physically block any binding of NEMO to IKK (Figures 3D

and 3E).

We next tested the specificity of NLRC5-mediated inhibition of

IKK phosphorylation. As shown in Figures 3H and 3I, NLRC5

markedly inhibited the IKKa/b phosphorylation, but not p38 or

JNK phosphorylation, consistent with the observation that

NLRC5 did not interact with TAK1 or MEKK3 (Figure S3A).

Finally, we further tested the status of the phosphorylation and

kinase activity of IKKa/b in IKKa/b /NLRC5 and IKKa/b /NEMO

complexes in RAW264.7 cells under physiological conditions.

IKKa/b in the IKKa/IKKb/NLRC5 (NLRC5-IP) complex was not

phosphorylated and showed no kinase activity during LPS

stimulation. By contrast, IKKa/b in the IKKa/b /NEMO (NEMO-IP)

complex was phosphorylated and exhibited a strong kinase

activity after LPS stimulation (Figure 3J), suggesting that NLRC5

inhibits its phosphorylation and kinase activity in the IKKa/

IKKb/NLRC5 complex under physiological conditions.


LRR Region Is Responsiblefor NLRC5-Mediated Inhibitionof IKK PhosphorylationTo identify the functional domains of

NLRC5, we generated four deletion con-

structs: NLRC5-D1, containing the CARD

and NOD domains (aa 1–517); NLRC5-D2,

containing the LRR-R1 (aa 651-898);

NLRC5-D3, containing the linker region

and LRR-R2 (aa 900–1329); and NLRC5-

D4, containing the CARD domain and

LRR-R3 (aa 1–215 plus 1471–1866) (Figure 4A). While all four

NLRC5 deletion constructs could interact with the full-length

IKKb protein as well as IKKb-KD (Figures 4B and 4C), NLRC5-

D3, like the full-length NLRC5, strongly inhibited NF-kB-luc

activity (Figure 4D). Other NLRC5 deletions showed either partial

or no inhibitory effect on NF-kB-luc activity. These results sug-

gest that NLRC5-D3 is required for the observed inhibition of

NF-kB activity by NLRC5.

To further investigate the molecular mechanism by which

NLRC5-D3 inhibits NF-kB activity, we tested whether these

deletions can inhibit the phosphorylation of IKKa/b. We found

that NLRC5-D3 strongly inhibited IKKa and IKKb phosphoryla-

tion, while NLRC5-D1, -D2 and -D4 produced little or weak inhi-

bition (Figure 4E). Notably, the inhibitory activity of these NLRC5

deletion mutants on the phosphorylation of IKKa and IKKb corre-

lated with their ability to inhibit NF-kB activation (Figure 4D), sug-

gesting that NLRC5-D3 inhibits NF-kB activation by blocking the

phosphorylation of IKK complexes. Unlike the full-length NLRC5,

however, NLRC5-D3 failed to compete with NEMO for binding to

IKKa/IKKb (Figure S4G), suggesting that the size of full-length

NLRC5 is critical for its ability to physically block NEMO from

interacting with IKKa/b.

Knockdown of NLRC5 Enhances NF-kB Activationas Well as the Inflammatory ResponseSince NLRC5 specifically interacts with IKKa/b and inhibits

NF-kB activation, we reasoned that knockdown of NLRC5 would

release IKKa/b for increased NF-kB activation under physiolog-

ical conditions. To test this prediction, we first demonstrated the

Figure 5. Knockdown of NLRC5 Can Signif-

icantly Enhance NF-kB Activation and

Inflammatory Responses

(A) Specific knockdown of NLRC5 or mNLRC5

was evaluated in various types of cells transfected

with NLRC5/mNLRC5 siRNA or scrambled siRNA.

(B) RAW264.7 cells were transfected with

mNLRC5 siRNA or scrambled siRNA, and then

treated with LPS. The cell extracts were harvested

at different time points and used for western blot

of various kinases and signaling proteins.

(C) The cell extracts of mNLRC5 knockdown and

control RAW264.7 cells after LPS treatment were

used for immunoprecipitation to obtain NEMO-

associated IKK (IP-1) and NEMO-free IKK (free

IKK and mNLRC5 associated IKK (IP-2). The phos-

phorylation of IKKa/b, the total amount of IKKa/b

and NEMO proteins in different fractions were

determined by anti-p-IKK, anti-IKKa/b or anti-

NEMO antibody.

(D) NLRC5 or mNLRC5 was knocked down in

293T/TLR4 and RAW264.7 cells. NF-kB-luc activ-

ity was determined after LPS treatment.

(E) The mNLRC5 knockdown and control AW264.7

cells were treated with LPS for 1 h; the nuclear

proteins were harvested for NF-kB binding activity

determined by EMSA. Oct-1 DNA-binding com-

plexes served as a control.

(F) Endogenous NLRC5 was knocked down in

THP-1 and RAW264.7 cells and TNF-a and IL-6

production was measured after LPS treatment.

Data in panels (D) and (F) are presented as

means ± SEM. Asterisks indicate significant

differences between groups (*p < 0.05, **p < 0.01,

***p < 0.001 as determined by Student’s t test

analysis).

See also Figure S5.

knockdown efficiency and specificity of NLRC5 or mNLRC5 at

both the mRNA and protein levels in various cell types with at

least two corresponding siRNAs, but not scrambled siRNAs

(Figure 5A and Figures S5A–S5C). We next tested the effect of

mNLRC5 knockdown on the phosphorylation of IKK, IkB, and

other kinases, including JNK, ERK, and p38. As shown in Fig-

ure 5B and Figure S5D, the phosphorylation of IKK (p-IKK) and

IkB (p-IkB) in the mNLRC5 knockdown cells was at least 3-fold

higher than that in the scrambled siRNA-transfected (control)

cells at 30 min after LPS treatment, although the total amounts

of IKK and IkB proteins were comparable between mNLRC5

knockdown and control cells. More importantly, we did not

Cell 141, 483–4

observe any appreciable differences in

p-JNK, p-ERK and p-p38 between

mNLRC5 knockdown and control cells,

clearly indicating the specific inhibitory

effect of mNLRC5 on the phosphorylation

of IKK, but not on JNK, ERK, and p38

phosphorylation. Similar results were

obtained with human THP-1 cells (Fig-

ure S5E). Collectively, these results sug-

gest that specific knockdown of NLRC5

or mNLRC5 strongly enhances the phos-

phorylation of the IKK complexes, but does not affect the phos-

phorylation of JNK, ERK, and p38 after LPS stimulation.

To further determine the molecular mechanisms by which

NLRC5 knockdown affects NF-kB signaling, we performed

two-step immunoprecipitations to obtain NEMO-associated IKK

(IP-1) and NEMO-free IKK (i.e., free IKK and NLRC5-associated

IKK, IP-2) (Figure 5C) and then compared the amounts of IKKa/

IKKb in each complex and their phosphorylation (p-IKK) in

mNLRC5 knockdown versus control cells. We found that the

total IKKa/b proteins in the NEMO-free IKK fraction (i.e., IP-2)

of mNLRC5 knockdown cells was slightly lower than that of

control cells, while IKKa/b in the IKKa/b/NEMO fraction (i.e.,

96, April 30, 2010 ª2010 Elsevier Inc. 489

IP-1) in mNLRC5 knockdown cells was slightly higher than that

in control cells, suggesting that the reduction of IKKa/b /NLRC5

complex in mNLRC5 knockdown cells decreases the IKKa/b

proteins in the NEMO-free IKK fraction, but increases the

IKKa/b proteins in the NEMO/IKK complex (Figure 5C). Con-

sistent with this observation, we found that the phosphorylation

of IKK (p-IKK) in the NEMO-containing IP fraction (IP-1) in the

mNLRC5 knockdown cells was higher than that in control cells.

Importantly, even though the total IKKa/b in the NEMO-free frac-

tion (IP-2) of mNLRC5 knockdown cells was less than that of

control cells, its phosphorylation (p-IKK) level in mNLRC5 knock-

down cells was higher than that in control cells (Figure 5C), sug-

gesting that mNLRC5 knockdown reduces the inhibition of phos-

phorylation of free IKKa/b. This notion agrees with data, showing

that NLRC5 interacts with constitutively active IKKa (EE) and

IKKb (EE) and inhibits their phosphorylation and kinase activity

(Figures S4D and S4E).

We next sought to determine whether the enhanced IKK phos-

phorylation seen with NLRC5 knockdown promotes NF-kB acti-

vation and NF-kB-dependent gene expression. Using the NF-kB-

luc reporter assay, we found that NLRC5 knockdown markedly

increased NF-kB-luc activity in 293T/TLR4 and RAW264.7 cells

after LPS treatment (Figure 5D). To directly demonstrate that

mNLRC5 knockdown enhances endogenous NF-kB activity,

we examined the DNA-binding activity of endogenous NF-kB.

Results in Figure 5E show that endogenous NF-kB activity in

mNLRC5 knockdown cells was at least 2-fold higher than that in

control cells. Consistent with this observation, knockdown of

NLRC5 or mNLRC5 resulted in markedly increased secretions

of NF-kB-responsive cytokines, such as TNF-a and IL-6, in both

mNLRC5 knockdown THP-1 and RAW264.7 cells (Figure 5F).

Hence, knockdown of NLRC5 or mNLRC5 enhances the IKKa/b

phosphorylation and NF-kB activity, thus increasing NF-kB-

dependent cytokine responses under physiological conditions.

NLRC5 Negatively Regulates Type I Interferon Signalingby Interacting with RIG-I and MDA5Recent studies show that NLRX1 inhibits the RIG-I-mediated

signaling pathway by targeting MAVS (Moore et al., 2008). To

determine whether NLRC5 might also be involved in the regula-

tion of type I interferon signaling, we performed functional assays

in TLR3-deficient 293T cells and found that intracellular poly(I:C)

activated IFN-b signaling, although such activation was strongly

inhibited by NLRC5 (Figure 6A), suggesting that NLRC5 func-

tions as a negative regulator of this antiviral pathway.

To determine the molecular mechanisms by which NLRC5

could inhibit the IFN-b response, we performed similar experi-

ments with different signaling molecules and found that RIG-I-

and MDA5-induced IFN-b-luc activities could be markedly

inhibited by increasing concentrations of NLRC5. However,

NLRC5 weakly inhibited MAVS- and TBK1-induced IFN-b-luc

activity and did not inhibit IKKi-induced IFN-b luciferase activity

(Figure 6A and Figure S6A). Furthermore, NLRC5 markedly

inhibited NF-kB-luc activity induced by intracellular poly(I:C),

RIG-I, MDA5, MAVS or TBK1 (Figure 6B). To determine whether

the weak inhibition of MAVS- and TBK1-induced IFN-b-luc activ-

ity by NLRC5 might be due to the inhibitory effect of NLRC5 on

IKK complexes, but not due to any direct effect on MAVS or


TBK1, we transfected 293T cells with ISRE-luc, together with

RIG-I, MDA5, MAVS, or TBK1 in the presence or absence of

NLRC5. Both RIG-I- and MDA5-induced ISRE-luc activities

were potently inhibited, while MAVS- and TBK1-induced ISRE-

luc activities were not (Figure 6C), suggesting that NLRC5 inhibits

IFN-b activation by directly interacting with RIG-I and MDA5, but

not with MAVS or TBK1. Indeed, we found that NLRC5 was

strongly associated with RIG-I and MDA5, but did not interact

with MAVS, IKKi, TBK1, TRIF, TRAF3, or IRF3 (Figures 6D and

6E and Figure S6B). Thus, NLRC5 specifically binds to the

RIG-I and MDA5 proteins to inhibit the IFN-b response.

To further define the interaction between RIG-I and mNLRC5

under physiological conditions, we infected RAW264.7 cells

with VSV-eGFP and performed immunoprecipitation with anti-

mNLRC5 at different time points after VSV-eGFP infection.

We detected only a weak RIG-I protein band in anti-mNLRC5 im-

munoprecipitants at 4 hr postinfection; however, it peaked at 6 hr

and then became weak at 8 and 10 hr postinfection (Figure 6F).

Notably, RIG-I protein expression was also increased at 6 hr

postinfection (Figure 6F), consistent with previous observations

(Honda and Taniguchi, 2006). Importantly, IRF3 phosporylation

was observed at 4 hr and further increased at 6 hr postinfection,

indicating that VSV-eGFP infection activated the IRF3-depen-

dent signaling pathway. However, IKK phosphorylation and IkB

degradation were weak after VSV-eGFP infection (Figure 6F).

These results suggest that interaction between NLRC5 and

RIG-I is inducible after VSV-eGFP infection.

Because both the RIG-I protein level and IRF3 phosphorylation

peaked at 6 hr postinfection, coincident with the peak interaction

between mNLRC5 and RIG-I in RAW264.7 cells, we considered

that this interaction might critically rely on activating signal.

To test this possibility, we generated RIG-I-CARD and RIG-I-heli-

case constructs and tested their ability to interact with NLRC5.

Interestingly, NLRC5 strongly bound to the CARD domain of

RIG-I, but not to the helicase domain, while binding of NLRC5

to the CARD domain of RIG-I markedly exceeded that to the

full-length RIG-I (Figure 6G), suggesting that the CARD domain

becomes accessible only when RIG-I is activated by virus-

derived ligands. Furthermore, competition experiments revealed

that NLRC5 strongly inhibited the ability of MAVS to bind to RIG-I

(Figure 6H). Thus, it appears that the accessibility of the CARD

domain of RIG-I is critical for a productive interaction between

NLRC5 and RIG-I, and that such interaction may also be influ-

enced by the relative concentrations of MAVS and NLRC5.

To confirm the inhibitory effect of NLRC5 on the IRF3 phos-

phorylation, we assessed the phosphorylation states of IRF3

by overexpressing NLRC5 in 293T cells and found that NLRC5

potently blocked the phosphorylation of endogenous IRF3

induced by RIG-I, but not by MAVS (Figure 6I). In contrast,

NLRC5 or mNLRC5 knockdown in THP-1 and RAW264.7 cells

markedly increased IRF3 phosphorylation (Figure 6J). Thus,

NLRC5 can inhibit IFN-b signaling by blocking the binding of

MAVS to RIG-I and the IRF3 phosphorylation.

Knockdown of NLRC5 Enhances Innateand Antiviral ImmunityTo further demonstrate the effects of NLRC5 knockdown on

the expression of IFN-responsive genes, we knocked down

Figure 6. NLRC5 Negatively Regulates IFN-b Activation by Inhibiting RIG-I and MDA5 Function

(A–C) 293T cells were transfected with NF-kB-luc, INF-b-luc or ISRE-luc, NLRC5 plus poly(I:C)/Lyovec, RIG-I, MDA5, MAVS, TBK1, or IKKi plasmids and analyzed

for INF-b or ISRE luciferase activity. Values are means ± SEM of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).

(D) 293T cells were transfected with HA-NLRC5 plus RIG-I, MAVS or IKKi. After immunoprecipitation with anti-HA beads, specific proteins were analyzed by

western blot with anti-Flag.

(E) 293T cells were transfected with MDA5 with or without HA-NLRC5. After immunoprecipitation with anti-HA beads, specific proteins were analyzed by western

blot with anti-MDA5.

(F) RAW264.7 cells were infected with VSV-eGFP, and cell extracts were harvested at different time points, immunoprecipitated with anti-mNLRC5 antibody and

analyzed by western blot with anti-RIG-I.

(G) NLRC5 binds to the CARD domain of RIG-I. 293T cells were transfected with HA-NLRC5 plus Flag-RIG-I, Flag-RIG-I CARD domain and Helicase domain (HD).

After immunoprecipitation with anti-Flag beads, specific proteins were analyzed by western blot with anti-HA.

(H) NLRC5 competitively binds to RIG-I with MAVS. HEK293T cells were transfected with Flag-MAVS-HA plus Flag-RIG-1, or Flag-NLRC5. After immunoprecip-

itation with anti-HA beads, specific proteins were analyzed by western blot with anti-Flag.

(I) 293T cells were transfected with Flag-RIG-I and Flag-MAVS, with or without HA-NLRC5, and used for western blot analysis with anti-phospho-IRF3 and IRF3

antibodies.

(J) RAW264.7 and THP-1 cells were transfected with NLRC5/mNLRC5-specific siRNA or scrambled siRNA, respectively, and then infected with VSV-eGFP.

The cell extracts were harvested for western blot with anti-phospho-IRF3 and IRF3 antibodies.

See also Figure S6.


Figure 7. Knockdown of NLRC5 Enhances Cytokine Response and Antiviral Immunity

(A and B) RAW264.7 cells or THP-1 cells were transfected with mNLRC5/NLRC5-specific siRNA or scrambled siRNA, followed by poly(I:C)/Lyovec treatment.

ISG-54, ISG-56, IFN-b mRNA and IFN-b protein were determined by real-time RT-PCR or ELISA.

(C) NLRC5 or mNLRC5 knockdown and control cells were infected with VSV-eGFP. Cell supernatants were used to measure IFN-b protein secretion by ELISA.

(D) RAW264.7 cells were transfected with mNLRC5 siRNA or scrambled siRNA for 36 hr and then treated with LPS, poly (I:C)/LyoVec (1 mg/ml) or VSV-eGFP

infection. Total RNAs from the treated cells were harvested at different time points and used for real-time PCR analysis to determine the expression of

TNF-a, IL-6, IFN-a, and IFN-b. The data in (A–D) are reported as means + SEM of three independent experiments. Asterisks indicate significant differences

between groups (*p < 0.05, **p < 0.01, ***p < 0.001 determined by Student’s t test analysis).


endogenous NLRC5 or mNLRC5 and then treated the cells with

poly(I:C)/Lyovec or infected them with VSV-eGFP. Real-time

PCR analysis revealed that poly(I:C)/Lyovec treatment or infec-

tion with VSV-eGFP strongly increased mRNA levels of IFN-b

and the interferon-stimulating genes ISG54 and ISG56 in cells

transfected with mNLRC5- or NLRC5-specific siRNAs (Figure 7A

and Figure S7A), consistent with a previous study showing the

upregulation of IFN-b mRNA in A549 cells with NLRC5 knock-

down (Opitz et al., 2006). Furthermore, we found that poly(I:C)/

Lyovec treatment or VSV-eGFP infection led to a large increase

in the production of IFN-b protein in THP-1, RAW264.7, primary

murine macrophages and primary human monocytes trans-

fected with mNLRC5 or NLRC5-specific siRNA (Figures 7B and

7C and Figure S7B). We next determined their expression

patterns over time after stimulation or VSV infection, and found

that the NF-kB-responsive genes TNF-a and IL-6 in mNLRC5-

knockdown cells were upregulated as early as 2–4 hr after LPS

treatment, and IL-6 continued in that state for another 10 hr

(Figure 7D). No difference was found between mNLRC5 knock-

down and control cells, with the exception of IFN-b expression

at 8 hr after LPS treatment (Figure 7D). Similar experiments

with intracellular poly(I:C) treatment or VSV infection showed

strong upregulation of IRF3-responsive IFN-a and IFN-b expres-

sion, but little or no effect on TNF-a and IL-6 expression

(Figure 7D). Consistent with these observations, we found that

LPS treatment resulted in more IL-6 and TNF-a than IFN-b

protein, while VSV-eGFP infection led to more IFN-b than TNF-a

and IL-6 proteins in mNLRC5 knockdown RAW264.7 cells

(Figure S7C). It appears that stimulation with LPS leads to

more pronounced effects on NF-kB-regulated genes than does

either poly(I:C) treatment or VSV-eGFP infection, while the

converse is seen for IRF-3- regulated genes.

To demonstrate a link between increased innate cytokine

responses and antiviral immunity in NLRC5-silenced cells, we

showed that knockdown of endogenous NLRC5 or mNLRC5

rendered cells remarkably resistant to viral infection and reduced

the levels of VSV-eGFP-positive cells among 293T, THP-1, and

RAW264.7 cells, as well as MEFs and human monocytes (Fig-

ure 7E and Figure S7D). To monitor VSV-eGFP propagation in

both mNLRC5 knockdown and control cells, we performed

time course experiments and showed that GFP expression

from VSV-eGFP could be observed at 8 hr postinfection with

rapid propagation at 10 hr in control cells. More than 90% of

the cells were GFP positive at 12 hr. By contrast, GFP expression

from VSV-eGFP viruses could rarely be observed at 10 hr,

became visible at 12–14 hr, and remained limited to a few cells

even at 18 hr in mNLRC5-silenced cells (Figure S7E). At 15 hr,

the morphology of the cells was markedly changed in control

cells, but not in mNLRC5 knockdown cells (Figure S7F). At 20 hr,

most of the cells in control group had died with a striking reduc-

tion in the GFP signal, while the cells treated with mNLRC5

siRNA remained alive and appeared normal (Figure S7G). We

conclude that NLRC5 knockdown can significantly increase

(E) 293T cells, THP-1 cells and RAW264.7 cells were transfected with NLRC5-, m

Viral infections were analyzed by fluorescence microscopy (with phase contrast

(F) Proposed model illustrating how NLRC5 negatively regulates both NF-kB and

See also Figure S7.

innate cytokine production and antiviral immunity against VSV-

eGFP infection and propagation.

DISCUSSION

Activation of innate immune receptors (TLRs, NLRs, and RLRs)

by their corresponding ligands initiates several key signaling

pathways, leading to the production of proinflammatory cyto-

kines, such as IL-6 and TNF-a, which in turn induce profound

positive feedback for adaptive immune responses (Akira et al.,

2006; Honda and Taniguchi, 2006). Increasing evidence indi-

cates that many inflammation-associated diseases may result

from dysregulated innate immunity (Inohara et al., 2005; Ting

et al., 2006). More recently, IL-1, IL-6, and TNF-a produced by

innate immune cells in chronic inflammation conditions have

been shown to promote cancer development and progression

(Karin et al., 2006). Thus, an understanding of the molecular

mechanisms by which innate immunity is held in check through

negative regulators appears critical for developing novel and

more effective treatments for inflammation-induced autoim-

mune diseases and cancer (Wang et al., 2008).

Both NF-kB and type I interferon signaling are controlled at

multiple levels by distinct mechanisms, whose regulatory pro-

teins may themselves be direct transcriptional targets of NF-kB

and type I interferon signaling, contributing to a negative regula-

tory feedback loop (Komuro et al., 2008). For example, expres-

sion of the A20, CYLD, and DUBA negative regulators is con-

trolled by NF-kB activity, while the RIG-I and MDA5 genes are

transcriptionally regulated by type I interferon signaling (Komuro

et al., 2008). We similarly observed the upregulation of NLRC5 or

mNLRC5 at both the mRNA and protein levels after 6 hr of treat-

ment with LPS. Such upregulation was abolished in MyD88-defi-

cient macrophages, suggesting that expression of NLRC5 itself

is under the control of MyD88-NF-kB pathways and forms

a negative regulatory feedback loop.

It has been demonstrated that the deubiquitinating enzymes

A20 and CYLD inhibit NF-kB signaling by targeting TRAF6

upstream of IKK (Kovalenko et al., 2003; Liew et al., 2005; Trom-

pouki et al., 2003; Wertz et al., 2004), while the deubiquitinating

protein DUBA inhibits type I interferon activity by targeting

TRAF3 (Kayagaki et al., 2007). Despite the importance of IKK

as a central transducer of signaling from cytokines, TLRs and

RLRs, leading to NF-kB activation, relatively little is known about

its negative regulation. Our findings show that NLRC5 blocks IKK

phosphorylation and thus NF-kB activation by interacting with

IKKa and IKKb, but not with the regulatory subunit NEMO.

To elucidate the mechanism(s) by which NLRC5 inhibits IKK

function, we provide evidence that NLRC5 forms a large com-

plex with IKKa/b, in addition to the previously described stable

IKKa/b/NEMO complex that seems to be dominant in unstimu-

lated cells (Rothwarf et al., 1998). The interaction between

NLRC5 and IKKa/b appears to be dynamic during the early

phase (1–2 hr) of LPS stimulation and correlates inversely with

NLRC5-specific siRNA or scrambled siRNA, and then infected with VSV-eGFP.

as a control) as well as FACS analysis.

type I IFN signaling pathways. Auto-p, autophosphorylation.


the phosphorylation of IKKa/b (p-IKK). This oscillation pattern

diminishes or even disappears with time after LPS stimulation,

suggesting the importance of this activating signal in the regu-

lation of the interaction between NLRC5 and IKKa/b. NLRC5

knockdown experiments showed increased cytokine responses

in NLRC5-silenced cells compared with control cells, suggesting

that the NLRC5 protein concentration is also an important fac-

tor in the regulation of the IKK activity. Overall, it appears that

both the LPS-induced activating signals and the relative protein

concentration of NLRC5 influence the interaction between

NLRC5 and IKKa/b, as well as the phosphorylation of IKK.

Although NLRC5 is ubiquitinated at about 30–40 min after LPS

stimulation (data not shown), further studies are needed to

determine whether the ubiquitination of NLRC5 plays a role in

the interaction between NLRC5 and IKKa/b.

A recent study shows that CUEDC2 interacts with IKKa/b, but

not NEMO, and recruits PP1c to deactivate the IKK complex

by dephosphorylating IKKa/b (Li et al., 2008). In resting cells,

CUEDC2 binds to IKKa/b (but not NEMO) and undergoes tran-

sient disassociation and reassociation steps after TNF-a treat-

ment (Li et al., 2008), similar to the interaction we described

between NLRC5 and IKKa/b after LPS stimulation. We also

show that IKKa/b in the IKKa/b /NLRC5 complex is not phosphor-

ylated and lacks kinase activity, in contrast to IKKa/b in the IKKa/

IKKb /NEMO complex (Figures 3E and 3J). These studies clearly

indicate that besides NEMO, other important regulatory proteins

such as NLRC5 can control the phosphorylation and kinase

activity of IKK complex through sequestering active IKKa/b.

Although the kinase domain of IKKb specifically recognized by

NLRC5 differs from the NEMO- binding site at the C terminus of

IKKb (May et al., 2000), we show that NLRC5 physically blocks

the binding of NEMO to IKKa/b due to its large size (Figure 3E

and Figure S4G). Our results also show that NLRC5 can interact

with constitutively active forms of IKKa (EE) and IKKb (EE) and

inhibit their ability to phosphorylate IkBa, as well as their ability

to autophosphorylate IKK (Figures S4D and S4E). Importantly,

experiments with two-step immunoprecipitation provide further

evidence that mNLRC5 knockdown has striking effects on IKK

and NF-kB signaling (Figure 5C), probably through a modulating

dynamic balance between /IKKa/b/NLRC5 and /IKKa/b/NEMO

complexes as well as sequestering the active IKKa/b to form an

inactive IKKa/b/NLRC5 complex. Furthermore, it has been sug-

gested that the phosphorylation of Ser740 in IKKb and Ser68 in

NEMO by active IKK may disrupt the interaction between IKK

and NEMO (Hayden and Ghosh, 2008), thus allowing other

proteins, including NLRC5 orphosphatases, to interact withactive

IKK and terminate NF-kB signaling. These studies suggest that

NLRC5 plays a critical role in inhibiting the phosphorylation and

kinase activity of IKKa/b in NF-kB activation after LPS stimulation.

RIG-I and MDA5 are key receptors for triggering type I inter-

feron signaling pathways, and are controlled by positive and

negative regulators, such as TRIM25 and RNF125, through ubiq-

uitination (Arimoto et al., 2007; Gack et al., 2007). To test the

hypothesis that other proteins are involved in the regulation of

RLRs, we present evidence showing that NLRC5 can bind to

both RIG-I and MDA5 but not to their downstream signaling

molecules such as MAVS, TBK1, IKKi, TRAF3, or IRF3. These

findings support a dual regulatory role for NLRC5 that encom-


passes both NF-kB and type I interferon signaling. Importantly,

our results show that NLRC5 binds more strongly to the CARD

domain of RIG-I than to the full-length RIG-I, suggesting that

NLRC5 specifically recognizes the CARD domain when it

becomes accessible after viral infection or stimulation by its

ligands. This interpretation agrees with the proposed model for

RIG-I action, in which the CARD domain of inactive RIG-I is

masked by its intramolecular binding with the repressor domain

in the C terminus (Cui et al., 2008; Yoneyama and Fujita, 2009).

Once RIG-I is activated, the CARD domain is exposed for

binding to MAVS or NLRC5.

Based on the experimental data discussed above, we propose

a model to illustrate how NLRC5 could negatively control both

NF-kB and type I interferon signaling (Figure 7F). First, the

expression of NLRC5 itself is controlled by NF-kB activation,

thus forming a negative regulatory feedback loop. Second, the

key to modulating the activation of NF-kB by NLRC5 in stimu-

lated cells lies in the dynamic balance between NLRC5/IKKa/

IKKb and NEMO/IKKa/IKKb complexes, which is controlled

by signaling stimulation and relative protein (NLRC5 versus

NEMO) concentrations, as well as the ability of NLRC5 to inhibit

IKK phosphorylation and kinase activity by sequestering active

IKKa/IKKb during signaling amplification after LPS stimulation.

Thus, the striking effects of NLRC5 on NF-kB signaling and its

downstream cytokine target genes cannot be explained by

only a small fraction of the NLRC5/IKKa/IKKb complex being

in an unstimulated state. Third, in contrast to its regulation of

NF-kB activation, NLRC5 competes with MAVS for binding to

the CARD domain of RIG-I or MDA5 only after it is exposed by

ligand stimulation of these receptors, leading to dampened

activation of IRF3. The interaction between NLRC5 and RIG-I

is inducible after poly (I:C) treatment or VSV viral infection.

Although TLR activation, cytokine stimulation and viral infection

can activate both NF-kB and type I signaling pathways, but they

tend to have a much more pronounced effect on either NF-kB-

regulated genes (e.g., LPS stimulation) or IRF3-regulated genes

(poly(I:C) or VSV infection). This selectivity appears to reflect the

different mechanisms by which NLRC5 regulates IKK activity or

RIG-I/MDA5 proteins. The ultimate outcome of NLRC5 inhibition

is determined by whether target gene promoters require NF-kB,

IRF3, or both transcription factors for gene expression.

Finally, we show that specific knockdown of NLRC5 not only

enhances NF-kB and type I interferon signaling and expression

of their target genes, but also increases antiviral immunity in

multiple cell lines and primary cells. Because of the conserved

biological function of NLRC5 in humans and mice, as well as in

various cell types, it appears to play a physiologically important

role in the maintenance of immune homeostasis, especially

with regard to regulation of the innate immune responses.

Hence, NLRC5 may provide a useful therapeutic target for

enhancing immunity against microbial infections and inflamma-

tion-associated diseases.

EXPERIMENTAL PROCEDURES

Molecular Cloning of Full-Length Human and Mouse NLRC5

A full-length NLRC5 cDNA was obtained from human PBMC cDNA by two-

step PCR and was then cloned into pcDNA3.1Z with HA tag sequence.

A similar strategy was used to clone mouse NLRC5. Both pcDNA-HA-NLRC5

and pcDNA-HA-mNLRC5 were sequenced to verify the correct DNA sequence

and their open reading frames.

Expression Profile and Antibody Production

The expression profile of NLRC5 and mNLRC5 in different tissues was evalu-

ated by reverse transcriptase (RT)-PCR analysis. NLRC5 and mNLRC5

peptides were used to generate polyclonal antibodies by standard methods.

Luciferase Assays, Immunoblot, Immunoprecipitation, and Kinase

Assay

HEK293 cells were transfected with IFN-b, NF-kB, or ISRE luciferase plasmids

and HA-NLRC5. TNF-a, IL-1b, LPS as well as exogenous MyD88, TRAF6,

IKKa, IKKb, NEMO, p65 (NF-kB), RIG-I, MDA5, MAVS, and IKKi plasmids

were used as stimulators. Dual-luciferase kits (Promega) were used for subse-

quent analysis. For kinase assay, a fusion of glutathione S-transferase and

amino acids of 1–54 of IkBa (GST-IkBa) was used as the substrate. To deter-

mine the kinase activity of immuoprecipitated IKKa (EE) and IKKb (EE) to

autophosphorylate IKK, we added 32p-ATP to immunoprecipitatants and

incubated the mixture at 30�C for 30 min. 32p-GST-IkBa (*p-GST-IkBa) and32p-IKK (*p-IKK) were detected by autoradiography.

Protein Fractionation by Size-Exclusion Column

Cell extracts prepared from RAW264.7 cell with lysis buffer were centrifuged at

20,000 rpm for 20 min at 4�C. Fifteen mg of protein in a volume of 1.5 ml was

loaded onto a size-exclusion column (HiPrep 16/60 Sephacryl S-300 HR) with

the capacity to separate the large protein complexes. Samples were fraction-

ated with a flow rate of 0.5 ml per min, and collected as 0.5 ml fractions after

passage through the void volume. Protein fractions were separated by SDS-

PAGE and detected by western blotting with antibodies against mNLRC5,

IKKa/b, or NEMO.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assays were performed by using the LightShift

Chemiluminescent EMSA kit from Pierce Biotechnology according to the

manufacturer’s standard protocol.

Real-Time PCR Analysis

First-strand cDNA was generated from total RNA using oligo-dT primers and

reverse transcriptase (Invitrogen). Real-time PCR was conducted with the

QuantiTect SYBR Green PCR Master Mix (QIAGEN) and specific primers on

an ABI Prism 7000 analyzer (Applied Biosystems).

Knockdown of NLRC5 and mNLRC5 by RNA Interference

NLRC5-specific, mNLRC5-specific and control (2-scramble mix) siRNA oligo-

nucleotides were obtained from Invitrogen and Integrated DNA Technologies,

and transfected into 293T, THP-1, RAW264.7 and primary cells with use of

Lipofactamine 2000 (Invitrogen) and various Nucleofector kits (one for each

cell type) according to the manufacturer’s instruction.

Statistical Analysis

The results of all quantitative experiments are reported as mean ± SEM of three

independent experiments. Comparisons between groups for Statistical Signif-

icance were performed with a two-tailed paired Student’s t test.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures and

seven figures and can be found with this article online at doi:10.1016/j.cell.

2010.03.040.

ACKNOWLEDGMENTS

We would like to thank Drs. Michael Karin and Bing Su for IKKa, IKKb, NEMO,

and p38 constructs; Yinling Hu and Feng Zhu for constitutively active IKKa/b

(EE) mutants and GST-IkB (1–54) constructs; Jae U. Jung for RIG-I construct;

Hongbin Shu for TRAF6; Kate Fitzgerald for TBK1 and IKKi constructs; and

John Hiscott for pISRE-luc construct. We would also like to thank Drs Shao-

Cong Sun, Xin Lin, and John Gilbert for their critical reading of this manuscript.

This work was supported in part by grants (P01CA094237, P50CA126752,

R01CA09327, R01CA101795, R01CA116408, and R01CA121191) from

National Cancer Institute, NIH, and Cancer Research Institute (to R.F.W).

J.C. and L.Z. were partially supported by NCET-06-0445 and the China Schol-

arship Council. Z.J.C. is an investigator of Howard Hughes Medical Institute.

Received: March 19, 2009

Revised: February 22, 2010

Accepted: March 25, 2010

Published: April 29, 2010

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figure 1. in vitro reconstitution of the rig-i pathway and...

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