The MAVS protein plays a critical role in the assembly of an antiviral signaling complex on mitochondrial membranes. Hou et al. (2011) now report that virus infection induces a conformational change in MAVS, leading to the prion-like formation of functional self-aggregates that provide a sensitive trigger for antiviral signaling

Maniatis, Tom

Ye, Junqiang

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Leading Edge
PreviewsA Prion-like Trigger
of Antiviral Signaling
Junqiang Ye1 and Tom Maniatis1,2,*
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
2Department of Biochemistry andMolecular Biophysics, Columbia University College of Physicians and Surgeons, NewYork, NY 10032, USA
*Correspondence: tm2472@mail.cumc.columbia.edu
DOI 10.1016/j.cell.2011.07.018
TheMAVSprotein plays a critical role in the assembly of an antiviral signaling complex onmitochon-
drial membranes. Hou et al. (2011) now report that virus infection induces a conformational change
in MAVS, leading to the prion-like formation of functional self-aggregates that provide a sensitive
trigger for antiviral signaling.Type I interferons (IFN a and b) play an
essential role in the cellular antiviral
response by coordinating the innate and
adaptive immune systems. Intracellular
viral RNA produced during virus infection
is detected by the RNA helicases RIG-I
orMDA5 (Skaug et al., 2009). RNA binding
induces a conformational change in these
helicases, exposing N-terminal tandem
caspase activation and recruitment
domains (CARDs) that bind to the CARD
domain in the adaptor protein MAVS,
which is located primarily on the outer
membrane of mitochondria (Seth et al.,
2005). MAVS associates with and signals
through adaptor proteins that activate
TBK1 and IkB kinases (IKK), which in
turn activate the transcription factors
IRF3/7 and NFkB, respectively. These
transcription factors translocate to the
nucleus and coordinately induce the
expression of IFN a and b and many other
antiviral genes. Many of the components
of this signaling cascade have been iden-
tified, and their role within the pathway
determined. However, the biochemical
mechanisms involved in signaling
complex assembly and the activation of
downstream signaling pathways are
poorly understood (Skaug et al., 2009).
In this issue of Cell, Hou et al. (2011)
exploit a robust and faithful in vitro system
for antiviral signaling (Zeng et al., 2009) to
uncover a remarkable prion-like mecha-
nism for signaling complex assembly.
Previous studies showed that activation
of RIG-I not only requires the binding of
double-stranded viral RNA, but also
binding of the CARD domain of RIG-I
to unanchored polyubiquitin chains of348 Cell 146, August 5, 2011 ª2011 Elsevierlysine-63 (K63) linkage (Zeng et al., 2010).
The Chen lab was the first to show that
covalently bound K63 ubiquitin functions
to mediate interactions between adaptor
proteins in the activation of IKK kinases
(Skaug et al., 2009). The RIG-I/ubiquitin
study reveals that short unanchored K63
ubiquitin chains are high-affinity ligands
that bind and activate RIG-I, thus ex-
panding the role of K63 ubiquitin in signal
transduction (Zeng et al., 2010).
Further exploitation of this system for
antiviral signaling now reveals an addi-
tional surprise—that virus infection
induces the formation of detergent-resis-
tant, well-ordered, high-molecular weight
aggregates of the MAVS protein, and
these aggregates are potent activators
of IRF3 (Hou et al., 2011). Recombinant
MAVS protein can form similar functional
aggregates in vitro. These aggregates
appear as amyloid fibril structures in the
electron microscope and are partially
resistant to proteinase digestion. Further-
more, preformed MAVS aggregates can
efficiently induce the endogenous MAVS
to form functional aggregates on themito-
chondria, suggesting that they are prion-
like. Additional experiments revealed
that the CARD domain of MAVS is both
necessary and sufficient for the formation
of MAVS fibrils in vitro. Strikingly, in the
presence of K63 ubiquitin chains and viral
RNA, RIG-I promotes the conversion of
monomeric MAVS proteins on the mito-
chondrial membrane to functional aggre-
gates that trigger downstream signaling.
A model for MAVS activation based on
the findings by Hou et al. is illustrated in
Figure 1.Inc.Prions are characterized by the patho-
genic properties of the so-called prion
protein PrP, associated with diseases
such as Creutzfeldt-Jakob disease in hu-
mans, and with the self-propagation of
multimers with properties of an amyloid
(Colby and Prusiner, 2011). This mecha-
nistic feature of prion aggregation has
also been observed with certain yeast
proteins and with the neuronal Aplysia
CPEB protein, all of which bear a gluta-
mine/asparagine Q-/N-rich domain (Si
et al., 2003). In the latter case, the neuro-
transmitter serotonin controls a prion-like
switch that results in a self-perpetuating
form that is proposed to mediate the
long-term maintenance of synaptic plas-
ticity and memory storage (Si et al., 2003,
2010). It is important to note that neither
CPEB nor MAVS display pathology or
transmission through generations. Si
et al. have proposed a new class of
proteins ‘‘that utilize a self-perpetuating
multimeric state to create a self-sustaining
altered activity state in the cell in response
to specific stimuli’’ (Si et al., 2010). The
MAVS protein fits this definition well, and
the mechanistic benefit is a high level of
sensitivity to virus infection. Remarkably,
fewer than 20 molecules of viral RNA and
K63 ubiquitin chains are sufficient to acti-
vate IRF3 (Zeng et al., 2010). A key unan-
swered question relates to the reversibility
ofMAVSconformational change.A ‘‘mem-
ory’’ of this change would not be a desired
property, as one may imagine this poten-
tially leading to uncontrolled antiviral
signaling and associated pathology.
It is important to note that there are no
known amino acid sequence similarities
Figure 1. MAVS Forms Prion-like Aggregates to Activate Innate Immunity
Virus infection activates the RIG-I/MAVS pathway. Viral RNA binds to the C-terminal regulatory domain
(RD) of RIG-I and induces a conformational change and dimerization. The central helicase domain is
required for this process. The CARD domain of the RIG-I dimer then binds unanchored lysine-63 (K63)
polyubiquitin chains and activates MAVS on the mitochondria membrane through the MAVS CARD
domain. This interaction triggers a conformational switch of MAVS (changed color and shape of MAVS
CARD in the diagram), which leads to the formation of MAVS aggregates on the mitochondrial membrane.
MAVS aggregates are composed of MAVS polymers and potently activate downstream signaling
cascade. Critical transcription factors IRF3 andNFkB are subsequently activated to induce the expression
of interferon and other antiviral genes. MAVS aggregates behave like prions: they are partially resistant to
detergent extraction and proteinase digestion and can efficiently self-propagate. The CARD domain of
MAVS directly mediates the self-aggregation, and the transmembrane domain (TM) greatly facilitates
MAVS aggregate formation on the mitochondria in vivo (left). Functional prion-like MAVS polymers can
also form by self-aggregation in vitro in the absence of the TM domain (right). MAVS polymers formed
in vitro can potently induce endogenous MAVS to form functional aggregates on the mitochondria from
uninfected cells. This property closely resembles that of infectious prions.between the MAVS polymers and the
glutamine/asparagine Q-/N-rich prions
or polyQ proteins. However, hints of
a possible alternative aggregate structure
are provided by a recent structural study
of Q-/N-rich and polyQ peptides showing
that they form a-helical coiled coils in vitro
and assemble into multimers (Fiumara
et al., 2010). Coiled coils (CCs) are known
to mediate protein-protein interactions
and oligomerization. A key role for this
structure in prion and polyQ expansiondiseases was provided by studies
showing that mutations that disrupt coiled
coils suppress aggregation and activity,
whereas mutations that enhance coiled
coils promote aggregation. Fiumara
et al. (2010) have proposed that CCs
may either be intermediate structures in
the transition from helical domains to
b sheets or they may be self-sufficient
mediators of the prion switch and amyloi-
dogenesis. As they point out, this mecha-
nism may have important implications forCell 1the distinction between pathogenic and
functional prions. Whereas the toxicity of
pathogenic prions and amyloids relies on
a misfolding into b sheets, nonpathogenic
and functional Q-/N-rich prions may use
CC-based conformational changes for
switching physiological activity on and
off in a regulated and persistent manner
(Fiumara et al., 2010). Whether this notion
applies to proteins lacking Q-/N-rich
sequences, such as MAVS, remains to
be established.
The structure of the MAVS CARD
domain has been solved and is character-
ized by a six-helical bundle arrangement
(Potter et al., 2008). The amino acid
sequences of the RIG-I/MDA5 and
MAVS CARD domains are only distantly
related; neither have obviousQ/N regions,
but both are essential for antiviral immu-
nity. Interactions between the CARD
domains of RIG-I/MDA5 and MAVS
promote the formation and spreading of
MAVS aggregates, which robustly acti-
vates downstream signaling. The MAVS
aggregates do not appear to be in the
form of a b-pleated sheet characteristic
of some prions based on the lack of stain-
ing with Congo red (Hou et al., 2011).
However, an interesting speculation is
that the helical bundles of the CARD
domain unfold into extended helices that
then interact as intermolecular coiled
coils.
Understanding the structural transition
of the MAVS CARD domains from the
monomer to the aggregated polymer
may provide insights into general mecha-
nisms for ordered aggregation-depen-
dent functional transitions. It seems likely
that the elegant mechanism for signaling
complex assembly proposed by Hou
et al. (2011) did not occur once in nature
and will soon be revealed in other
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315–330.Regulatory Revolution:
Evolving the ‘‘Anti-LacI’’ Repressor
Christopher J. Marx1,2,*
1Department of Organismic and Evolutionary Biology
2Faculty of Arts and Sciences Center for Systems Biology
Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
*Correspondence: cmarx@oeb.harvard.edu
DOI 10.1016/j.cell.2011.07.015
Much of adaptation is based upon changes in gene expression, but the emergence of new regula-
tory logic has not been observed directly. Now, Poelwijk et al. report evolving the lac repressor
(LacI) to reverse its regulatory logic, resulting in an ‘‘anti-LacI’’ that represses transcription when
bound to its ‘‘inducer.’’From the origin of new body-plans
(Carroll, 2008) to beneficial changes in
beak morphology (Mallarino et al., 2011),
the evolution of gene regulation is critical
in adaptation over many timescales.
Evenwhen populations ofmicrobes adapt
in the laboratory, promoters and regula-
tory proteins are disproportionately
recovered as targets of beneficial muta-
tions (Barrick et al., 2009). The selective
advantage of experimentally evolved
changes in expression has typically arisen
from altered levels of the affected gene
products, but what about the evolution
of new regulatory logic itself?
Most laboratory experiments use a
constant environment for selection, and
thus, developing new responses to
changes in the environment tend not to
be particularly advantageous. Further-
more, it is unclear how many mutations,
perhaps acting in concert, are needed to
change the qualitative properties of a
gene regulatory system. In this issue,
Poelwijk et al. present a tour de force study
in which they use an elegant synthetic
system to apply alternating selective pres-
sures on a genetic module of Escherichiacoli (Poelwijk et al., 2011). In the end, the
authors coerce the E. coli lactose re-
pressor (encoded by lacI) to have exactly
the opposite logic as it does naturally.
The E. coli lactose repressor, encoded
by the lacI gene, is one of the best under-
stood transcriptional regulators. It binds
the lac operator and represses trans-
criptional initiation in the absence of its
inducer such as allolactose or its ana-
log isopropyl b-D-1-thiogalactopyrano-
side (IPTG), but once LacI binds IPTG,
the repressor releases from the operator
(Figure 1A).
In order to select for the opposite
response, that is repression in the pres-
ence of IPTG and lack thereof in its
absence, Poelwijk et al. construct an
operon with the lac operator controlling
two genes that permit expression to be re-
warded (by resistance to the antibiotic
chloramphenicol) or punished (by sensi-
tivity to sucrose) depending upon which
chemical is added to the medium. Poel-
wijk et al. also include in the operon the
reporter gene lacZ, which encodes
b-galactosidase, to quantify total expres-
sion of the operon.From here, the strategy is simple. First,
iteratively introduce a hearty dose of
variation specifically in lacI by PCR muta-
genesis. Next, select upon this variation
by alternating periods of growth in me-
dium supplemented with sucrose and
IPTG with growth in medium with chlor-
amphenicol and not IPTG (i.e., to select
for the opposite logic). Then, repeat.
What are the chances that LacI would
readily reverse its response to inducer
and become the ‘‘anti-LacI’’? After the
first round of variation and selection, the
results did not seem promising. In fact,
the evolution appears to reach a road-
block. LacI variants that abandon the
wild-type logic (which led to maximal
punishment emerged) exhibit no logic at
all; they simply allow constitutive expres-
sion of the downstream genes, com-
pletely independent of IPTG (Figure 1A).
After the next round, however, Poelwijk
and colleagues find variants that perform
the desired inverse logic, and these vari-
ants dominate after the final, third round.
The authors then carefully analyze the
genetic basis of this adaption. Reversing
the logic of the Lac repressor is indeed


A Prion-like Trigger of Antiviral Signaling 

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A Prion-like Trigger of Antiviral Signaling

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