n e w s a n d vi e w s

Breaking the barrier: membrane fusion triggers innate

antiviral immunity
David Olagnier & John Hiscott

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2012 Nature America, Inc. All rights reserved.

The sensing of viral infection by the innate immune system is dominated by the recognition of nucleic acids.
New data now demonstrate that the fusion of viral and target-cell membranes leads to the activation of an immune
response dependent on the adaptor STING.

he innate immune system has evolved

many molecular sensors and signaling
pathways to activate host-defense mechanisms that counteract viral infection1. Cells
of the immune response sense viral infection
largely through germline-encoded patternrecognition receptors (PRRs) that recognize
evolutionarily conserved structures known
as pathogen-associated molecular patterns.
Classically, viral nucleic acids are the predominant pathogen-associated molecular patterns
detected by these receptors during infection,
although in some cases viral proteins can function as ligands for PRRs1. These sensing events
signal the production of type I interferons as
a host-intrinsic antiviral defense. In this issue
of Nature Immunology, Holm et al. describe a
previously unknown mechanism of recognition by the innate immune system mediated by
the fusion of viral and target-cell membranes,
which occurs independently of the sensing
of viral DNA or RNA2. This fusion process
activates a signaling pathway dependent on
the adaptor STING (MITA, MYPS or ERIS),
which leads to the specific production of type I
interferons and expression of interferonstimulated genes (ISGs)2.
Among the PRRs, a subgroup of Toll-like
receptors (TLRs) located in the endosome
sense viral nucleic acids and signal through
the recruitment of adaptors such as MyD88
and/or TRIF3. Distinct from TLR-dependent
sensing, viral RNA structures in the
cytoplasm are recognized by members of the
RIG-I-like receptor (RLR) family of PRRs,
David Olagnier and John Hiscott are in the Division
of Infectious Diseases, Vaccine & Gene Therapy
Institute of Florida, Port St. Lucie, Florida, USA.
e-mail: jhiscott@vgtifl.org

which includes the three DExD/H-box RNA

helicases RIG-I, Mda5 and LGP-2 (ref. 4).
Sensors of viral DNA such as DAI or AIM2
are also involved in these virus-sensing processes5. In addition, the sensors LRRFIP1
and IFI16 have been suggested to be intracellular regulators of DNA-driven signaling
by the immune system5. Also in the family
of DexD/H-box RNA helicases, DHX36
has been shown to recognize CpG DNA in
plasmacytoid dendritic cells, which leads to
activation of the transcription factor NF-B5.
Although the recognition of cytoplasmic
DNA is linked to the transcriptional induction of type I interferons and proinflammatory cytokines, cytosolic DNA also mediates
the activation of caspase-1 and maturation of
the proinflammatory cytokines interleukin
1 (IL-1) and IL-18, which links the inflammasome to innate antiviral immunity6.
In the present study, Holm et al. describe a
previously unknown element of detection by
the innate immune system that is dependent on
the fusion process that occurs between the viral
envelope and cellular membrane but is independent of the sensing of viral DNA or RNA2.
The authors generate virus-like particles (VLPs)
derived from herpes simplex virus type 1
(HSV-1) that lack capsid and nucleic acid but
are able to induce a type I interferon response
in both primary mouse cells and primary
human cells2. In contrast to fusogenic VLPs,
HSV-1-derived VLPs that lack fusogenic ability because of absence of the fusogenic glyco
proteins gB or gH on the VLP are unable
to induce type I interferon or ISGs, which
indicates that the fusion process itself represents the danger signal detected by the innate
response. The sensing phenomenon is not
restricted to infectious contexts, because they

nature immunology volume 13 number 8 auGust 2012

also observe similar triggering of the type I

interferon response after cell-cell fusion or
liposome-cell fusion; thus, this demonstrates
a previously unknown membrane fusion
activated pathway that leads to type I interferon
responses. In fact, some aspects of these results
were portended by published work demon
strating that membrane perturbations induced
by cell-cell fusion are sufficient to elicit an
antiviral state and ISG induction7. However,
there is an important distinction between
these two studies: Holm et al. use cells from
mice deficient in the -chain of the receptor
for interferon- (IFN-) and IFN- to demonstrate that the induction of ISGs in response to
the fusion process is dependent on the production of type I interferons2, whereas the earlier
study indicates that membrane perturbation
induces a subset of ISGs in the absence of such
interferon production7. Both studies highlight
the fusion process as a key element of detection by the innate immune system but differ in
their conclusions about the role of type I interferons in the process. The specific function of
the type I interferon response in infectious and
noninfectious fusion processes will require
further attention.
Holm et al. also assess whether the production of IFN- and the chemokine CXCL10
generated by membrane fusion is dependent
on known virus-sensing pathways2. In studies
of cells from mice deficient in various adaptors
(MyD88 and/or TRIF for TLRs; MAVS for RLRs;
and STING for DNA sensors), they observe
that the induction of CXCL10 in response to
treatment with VLPs or liposomes is abolished
only in cells from STING-deficient mice2.
STING is an endoplasmic reticulumresident
transmembrane protein previously demonstrated to exert a potent antiviral effect through
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n e w s an d v i e w s
Type I IFN
IFNAR1
PLC-

ROS
Ca2+

PI(3)K

Pro-IL-1
Akt

IB
p50 p65

ASC

STING

Cytosolic NLR

Katie Vicari

2012 Nature America, Inc. All rights reserved.

npg

Tyk2

STAT1
STAT2

IRF9

TBK1

STAT1
IRF9
STAT2 P

IRF3

Inflammasome
complex

No proinflammatory
response

Cytoplasm

Type I IFN

IRF3
IRF3

STAT1
STAT2 P

Endoplasmic
reticulum

NF-B

Pro-caspase1

Jak1

IFNAR2

Nucleus
P Ifnb1

Tnf

IRF3
IRF3
P

ISGs
P
STAT1
IRF9
STAT2 P

IFN antiviral
state

Figure 1 Fusion of the viral membrane with the host-cell membrane triggers an intrinsic antiviral
response that is dependent on STING. Membrane fusion activates the PLC-PI(3)K pathway and
stimulates the release of Ca2+ from the endoplasmic reticulum. The response to membrane fusion
is restricted to a type I interferon response that depends on an intact STING-TBK1-IRF3 pathway,
with limited effects on the expression of genes encoding inflammatory molecules or inflammasome
activation. IFNAR1 and IFNAR2, receptors for type I interferon; Akt, Jak1 and Tyk2, kinases; IB,
inhibitor of NF-B; p50 and p65, subunits of NF-B; ASC, adaptor; NLR, pattern-recognition receptor;
Ifnb1, gene encoding IFN-; Tnf, gene encoding tumor-necrosis factor.

the production of type I interferons810. As

STING has been shown to relocalize from the
endoplasmic reticulum to perinuclear vesicles
together with the kinase TBK1 after HSV-1
infection10, Holm et al. investigate the intra
cellular localization of STING after treatment
with VLPs (or liposomes) and find that 50%
of treated cells demonstrate relocalization of
STING and colocalization of STING with TBK1
(ref. 2). Studies of cells from Tbk1/ and Irf3/
mice show that the transcription factor IRF3
is essential in promoting CXCL10 production
in response to VLPs and liposomes, whereas
TBK1 is involved but not essential in this, as
shown by the lower but detectable expression
of CXCL10 by VLP-stimulated Tbk1/ cells.
This result suggests a more-complex signaling
pathway downstream of STING that leads to
IRF3 activation, rather than the classic STINGTBK1-IRF3 pathway involved in the sensing of
viral DNA5.
Infection with HSV triggers the activation
of calcium-dependent pathways as an early
signaling event after membrane fusion11.
Consistent with that observation, Holm et al.
demonstrate that membrane fusion is linked to
activation of the phospholipase C- (PLC-)
phosphatidylinositol-3-OH kinase (PI(3)K)
714

pathway and to the release of free intracellular Ca2+ from endoplasmic reticulum stores2.
Furthermore, chemical inhibitors of PLC- and
PI(3)K block the VLP-induced increase in the
release of free intracellular calcium and expression of CXCL10. These data indicate that the
PLC-PI(3)K pathway has a role in stimulating a STING-dependent immune response.
Proteins such as the membrane phospholipid
PLSCR1 that are involved in the modulation
of membrane dynamics in response to higher
calcium concentrations have been shown to
potentiate the antiviral activity of interferons
by increasing the expression of genes encoding
antiviral proteins12. Holm et al. assess whether
PLSCR1 is involved in fusion-dependent signaling by using Plscr1/ cells but find that it
is not involved in the expression of ISGs in
response to membrane fusion2. Although
PLC- is classically activated by G protein
coupled receptors, dynamic reorganization of
the membrane structure after fusion may be
sufficient to induce relocation of PLC- into
lipid membrane rafts and activation independently of G protein coupledreceptors. The
mechanistic link between the PLC-PI(3)K
pathway and assembly of the STING-TBK1
complex that leads to IRF3 activation is not

explored further, and studies to ascertain

whether the PLC-PI(3)K pathway and release
of Ca2+ are directly involved in activation of the
STING-TBK1-IRF3 pathway are warranted.
A surprising outcome of this study is the
observation that the innate response to membrane fusion is restricted to a type I interferon
response, with a limited effect on the expression of genes encoding inflammatory proteins,
inflammasome activation or autophagy2.
Interestingly, treatment with VLPs also synergistically enhances the responses of TLR7
and TLR9 to their respective agonists, which
suggests that membrane fusion optimizes
the innate response to viral nucleic acids. The
mechanistic basis of this effect is unknown,
although the generation of reactive oxygen
species (ROS) may be involved in this TLR
priming process. Indeed, a published study
has demonstrated that infection with HSV
increases ROS production and potentiates signaling from PRRs13; by extension, HSV-derived
VLPs seem to serve a similar role in stimulating
ROS production. ROS production represents
a potent danger signal for activation of the
inflammasome14 and NF-B15. Surprisingly,
IL-1 release, inflammasome activity and
production of tumor necrosis factor are not
detected by Holm et al. after membrane fusion,
despite ROS production2. Membrane fusion is
thus the first example of an innate recognition mechanism that selectively activates the
interferon response. However, further studies
are needed to elucidate how the fusion process
selectively triggers the interferon response
without affecting proinflammatory signaling.
For example, is the magnitude of ROS production associated with membrane fusion insufficient to trigger inflammatory pathways? Is
ROS production incompatible with the localization of proinflammatory activating factors?
These issues should be addressed to delineate
how membrane fusion specifically triggers a
STING-dependent type I interferon response.
With the present study, Holm et al. have
identified a previously unknown, nucleic
acidindependent mechanism of sensing
viral infection that is mediated by the fusion
of viral membranes and cellular membranes2.
This fusion process selectively induces a
type I interferonISG response through a pathway dependent on STING and involving TBK1,
IRF3 and the PLC-PI(3)K (Fig. 1). This
pathway is not restricted to the infectious context, as fusion-dependent sensing also occurs
in response to liposome-cell fusion or cell-cell
fusion. However, it remains to be determined
how this pathway may distinguish a potentially
dangerous microbial infection from innocuous physiological fusion events such as endosome fusion, syncytia formation and so on.

volume 13 number 8 auGust 2012 nature immunology

n e w s an d v i e w s
This study demonstrates that disrupting membrane integrity during the very initial stages
of virus infection represents another danger
signal sensed by the cell-intrinsic response. The
activation of a STING-dependent pathway via
PLC-PI(3)K adds another layer of recognition and response available to the cell to counteract invasion by viruses and microbes.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.

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(2012).
3. Kawai, T. & Akira, S. Immunity 34, 637650
(2011).
4. Loo, Y.M. & Gale, M. Jr. Immunity 34, 680692
(2011).
5. Keating, S.E., Baran, M. & Bowie, A.G. Trends Immunol.
32, 574581 (2011).
6. Rathinam, V.A. et al. Nat. Immunol. 11, 395402
(2010).
7. Noyce, R.S. et al. J. Virol. 85, 1092610931
(2011).

8. Zhong, B. et al. Immunity 29, 538550 (2008).

9. Ishikawa, H. & Barber, G.N. Nature 455, 674678
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12. Dong, B. et al. J. Virol. 78, 89838993 (2004).
13. Gonzalez-Dosal, R. et al. PLoS Pathog. 7, e1002250
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Hair follicles: gatekeepers to the epidermis

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2012 Nature America, Inc. All rights reserved.

William R Heath & Scott N Mueller

Substantial depletion of Langerhans cells leads to their replenishment by bone marrowderived precursors that
access the epidermis through hair follicles, a site of crucial chemokine production.

angerhans cells are an archetypal population of dendritic cells that reside in the
outer layer of the skin, monitoring for infectious agents, which they capture and transport
to the draining lymph nodes and then present
to T cells1. Although they are derived mainly
by self-renewal, when conditions lead to depletion of Langerhans cells, such as after exposure to ultraviolet light, these dendritic cells
can be replenished by bone marrowderived
monocytes recruited from the blood2. In this
issue of Nature Immunology, Nagao and colleagues show that access of precursors of
Langerhans cells to the epidermis is not random
but depends on hair follicles3. These complex
structures not only provide a conduit for access
to the outer layer of the skin but also supply
a range of chemokines necessary for attracting precursor cells to sites of depletion of
Langerhans cells. This study raises the intriguing possibility that hair follicles act broadly as
gatekeepers for the epidermis.
The skin is a complex organ with many
roles in everyday physiology, including acting
as a sensory organ, a barrier to the external
environment, a seal to prevent dehydration,
a temperature control system and a first line
immunological defense via various innate and
adaptive mechanisms. Mammalian skin consists of many layers, of which the epidermis is
the outer layer; it is only a few cells thick in
mice but is considerably thicker in humans

William R. Heath and Scott N. Mueller are in the

Department of Microbiology and Immunology,
The University of Melbourne, Parkville, Australia.
e-mail: wrheath@unimelb.edu.au or
smue@unimelb.edu.au

and is covered by a dead cornified epithelium. That cornocyte layer, together with various secretions and a superficial live stratum
granulosum layer of keratinocytes sealed by
tight junctions, offers a stringent and waterproof
barrier to the outside environment while still
enabling the secretion of various fluids and oils
through pores and hair follicles as well as access
of dendritic cells to external antigens through
regulated separation of keratinocyte tight junctions. Below the epidermis, and separated by a
basement membrane, lies the dermis, which is
thicker than the epidermis and is a less-dense
cellular network that incorporates various
cell types into a complex extracellular matrix
composed mainly of collagen. The dermis is
invaded by fine capillaries and nerve endings
and is penetrated by hair follicles that span the
entire epidermis. Underneath those two cutaneous layers is subcutaneous tissue composed
largely of fat, collagen and fibroblasts.
Although keratinocytes make up the bulk of
the epidermis, there are many other important
cell types associated with this layer, including
melanocytes, which provide protection against
exposure to ultraviolet light; merkel cells,
which detect light touch; T cells, which in
the mouse seem to function in tissue repair;
and Langerhans cells, a type of dendritic cell
distributed in a dense network extensively
throughout the epidermis. Those last cells
sample antigens associated with the skin and
transport them to the draining lymph nodes for
presentation to T cells. The role of Langerhans
cells in initiating skin-associated immunity is
controversial, although emerging evidence suggests that they participate in responses to some
fungal and bacterial infections and may contribute to tolerance induction4,5. The dermis

nature immunology volume 13 number 8 auGust 2012

contains at least two other types of dendritic

cells that are also able to initiate immunity to
skin infections. Although these dermal dendritic cells are clearly of bone marrow origin
and are replenished by bone marrowderived
precursors after lethal irradiation, the origin
of Langerhans cells is more complex. Under
steady-state conditions, most Langerhans cells
self-renew2,6. This differs from the renewal of
other dendritic cell populations, which are
renewed mainly from bone marrowderived
precursors. However, Langerhans cells can
be replenished from bone marrowderived
monocytes2, especially after depletion of the
bulk of these cells from the skin. After entering
epidermis replete in Langerhans cells, monocytes rapidly upregulate major histocompatibility complex (MHC) class II molecules and
convert into true Langerhans cells.
Nagao and colleagues use complex bone
marrow chimeras to enable specific depletion
of Langerhans cells from normal skin by the
administration of diphtheria toxin3 (Fig. 1).
They then examine the replenishment of
these cells under steady-state conditions or
after various inflammatory stimuli. They
confirm published work suggesting that skin
lacking Langerhans cells in adult mice can be
repopulated by monocytes2, but they also note
the involvement of a second precursor cell.
Although the monocyte-derived precursor
first appears in the epidermis as a langerinnegative, MHC class IIpositive, CD11b+ cell,
the nature of the second precursor cell is
unknown, and its existence is inferred only
from the presence of fully formed Langerhans
cells that are not derived from transferred
monocytes. Perhaps this second precursor cell
actually seeds the hair follicles of the epidermis
715