how viruses manage to control TLR pathways

how viruses manage to control TLR pathways

Briefly describe how viruses manage to control TLR pathways. For instance, how does a dsRNA activate the TLR pathway compared to ssRNA. Utilize multiple viruses as examples and focus on the differences between cell types that are regulated in a TLR dependent manner vs. other cells that are not.
Briefly describe how viruses manage to control TLR pathways. For instance, how does a dsRNA activate the TLR pathway compared to ssRNA. Utilize multiple viruses as examples and focus on the differences between cell types that are regulated in a TLR dependent manner vs. other cells that are not.
please include Tat transactivation factor for HIV and viral progression in the answer.
please use only Pubmed database as a reference (5 different sources) and no other reference data base. And do in text citation.
Review
Innate immune recognition of DNA: A recent history

abstract
Innate immune DNA sensing underpins many physiological and pathological responses to DNA,
including anti-viral immunity to DNA viruses. Although it has been appreciated for many years that
cytosolic DNA can evoke a type I interferon response, it is only within the past decade that the cellular
mechanisms responsible for such a response have been defined. Here we review the discoveries that led
to an appreciation of the existence of cytosolic DNA sensor proteins, and discuss two key such sensors,
cGAS and IFI16, in detail. DNA sensors operate via STING, a protein shown to have a central role in
controlling altered gene induction in response to DNA in vivo, and as such to be central to a rapidly
expanding list of both protective and harmful responses to DNA. We also discuss recent insights into
how and when DNA stimulates innate immunity, and highlight current outstanding questions in the
DNA sensing field.
& 2015 Elsevier Inc. All rights reserved.
Contents
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Early studies in defining cytosolic DNA sensor signaling (2006–2009) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
The centrality of STING in controlling DNA sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Discovery of multiple putative DNA sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
Mechanisms of innate DNA sensing: lessons from PYHIN proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Discovery of cGAS as a critical DNA sensor acting via STING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Introduction
Innate immunity has undergone a revolution in the past 15 years,
with the uncovering of the molecular basis underpinning Charles
Janeway’s now validated hypothesis of the existence of germline
encoded pattern recognition receptors (PRRs) capable of detecting
and responding to pathogen-associated molecular patterns (PAMPs)
on microbes (Janeway, 1989). With the discovery of the Toll-like
receptors (TLRs), mechanisms for pathogen detection, leading in
particular to induction of cytokines and type I interferons (IFN-Is),
were revealed, and thus the innate immunity renaissance began
(O’Neill et al., 2013). In terms of viral detection, cell membrane bound
TLRs were shown to recognize glycoproteins on the surface of viral
capsids, while endosomally placed TLRs responded to viral nucleic
acid, in particular viral RNA (O’Neill et al., 2013). Rapid progress was
made from 2004 in understanding how PRRs also survey and protect
the cytosolic compartment from viruses, with the discovery and
characterization of the RIG-I-like receptors (RLRs) RIG-I and MDA5,
which recognize viral RNA genomes, replication intermediates and/or
transcription products (Yoneyama et al., 2004; Andrejeva et al.,
2004). Further structural studies of RLRs led to an appreciation of
how these PRRs distinguish self from non-self RNA, in that RIG-I is
activated by viral RNA with a 50
triphosphate, or a 50
diphosphate,
moiety juxtaposed to a dsRNA panhandle, while MDA5 binds to long
dsRNA (Gurtler and Bowie, 2013; Goubau et al., 2014). Both RLRs then
transduce a signal via MAVS, leading to activation of transcription
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
Virology
http://dx.doi.org/10.1016/j.virol.2015.03.013
0042-6822/& 2015 Elsevier Inc. All rights reserved.
n Corresponding author. Fax: þ353 1 6772400.
E-mail address: agbowie@tcd.ie (A.G. Bowie).
Virology 479-480 (2015) 146–152
factors such as NF-κB and IFN regulatory factor 3 (IRF3) and
subsequent cytokine and IFN-I induction.
In light of these discoveries and in the context of the paradigm of
PRRs recognizing PAMPs, the questions of how, why and when DNA
activates the innate immune system has received considerable
attention in the past decade. The immunostimulatory and anti-viral
potential of DNA introduced into mammalian cells had been reported
more than 50 years ago (Isaacs et al., 1963; Rotem et al., 1963), but
only recently have the cellular mechanisms of DNA detection leading
to immune responses, and to IFN-I induction in particular, been
identified. This has led to an understanding of the role of cytosolic
DNA sensing in many protective and pathological responses to DNA,
for example in detection of DNA viruses and in aberrant responses to
self-DNA leading to autoimmunity respectively.
Here we review the discovery of proposed cytosolic DNA sensors
over the past decade (Fig. 1), discuss the current understanding of
Fig. 1. Timeline for discovery of putative cytosolic DNA sensors and associated signaling molecules. The chronology of the identification of intracellular DNA sensors is
shown. DNA sensors are in red, key signaling proteins in green, and the RNA sensors Rig-I and MDA5 shown for comparison (orange). As detailed in the text, certain DNA
sensors (boxed in gray here) have been confirmed to have a role in DNA- and/or DNA-virus dependent cytokine and IFN-I production in vivo, by using knockout mice.
Fig. 2. Cytosolic DNA sensors and their signaling pathways. Upon recognition of dsDNA in the cytosol or nucleus, DNA sensors signal via adaptor proteins to alter gene
induction via transcription factors (IRFs and NF-κB), and to pro-IL-1β processing, via Caspase 1. DNA sensors described in the main text are shown in red.
A. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152 147
how such sensors operate to respond to immune-stimulatory DNA
to mobilize immune responses (as outlined in Fig. 2), and highlight
current issues that remain to be resolved in this rapidly evolving field.
Early studies in defining cytosolic DNA sensor signaling
(2006–2009)
The first PRR implicated in detection of DNA was TLR9 (Fig. 1),
shown in 2000 to be responsible for the ability of mice to respond
to CpG DNA (Hemmi et al., 2000), leading to cytokine and IFN-I
induction via NF-κB and IRF7 respectively (O’Neill et al., 2013).
This provided an elegant example of self-versus non-self immune
discrimination, since TLR9 recognized under-methylated DNA
(i.e. CpG DNA), which is enriched in microbial genomes compared
to mammalian cells. In contrast, it is now clear that if either self or
pathogen dsDNA accumulates in the cytosol, TLR9-independent
cytokine and IFN-I induction is elicited in many cell types (reviewed
in (Paludan and Bowie, 2013)). TANK-binding kinase (TBK1) was
identified in 2006 as being required for IFN-β production in response
to transfected synthetic dsDNA (poly(dA:dT)) (Ishii et al., 2006)
(Fig. 1). After activation of upstream PRR signaling, TBK1 directly
phosphorylates IRF3 leading to IFN-β induction, and the TBK1-IRF3
signaling axis was quickly established as fundamental to the cytosolic
DNA response to other forms of synthetic dsDNA (Stetson and
Medzhitov, 2006), to DNA viruses and for the immune response to
DNA vaccines (reviewed in (Keating et al., 2011)). Thus by 2006
the search was on to discover one or more DNA sensors that would
directly bind to cytosolic DNA and mediate IFN-I induction via a
signaling pathway involving TBK1 and IRF3.
In 2007, the first reported cytosolic DNA receptor was identified
as DNA-dependent activator of IRFs (DAI) (Takaoka et al., 2007). DAI
(or ZBP-1), encoded by an IFN-inducible gene, was found to be
capable of upregulating the expression of IFN-I via NFκB and IRF3 in
response to poly(dA:dT), and to bind to DNA (Takaoka et al., 2007).
DAI knockdown in mouse L929 cells reduced HSV-1-stimulated IFN-
β production, while subsequent studies showed a role for DAI in IFN-I
and cell death responses to CMV in fibroblasts (DeFilippis et al., 2010;
Upton et al., 2012, 2010). However, no role for DAI in macrophage
responses to DNA was observed (Unterholzner et al., 2010), while the
in vivo response to DNA vaccination was TBK1-dependent, but DAIindependent
(Ishii et al., 2008). This indicated the existence of other
as of yet unidentified cytosolic DNA sensors, especially for myeloid
cells. A further DNA sensing pathway was identified in 2009, which,
surprisingly for a DNA response, involved RIG-I and MAVS (Ablasser
et al., 2009; Chiu et al., 2009). It was shown that RNA polymerase III
(RNA Pol III) is capable of transcribing AT-rich dsDNA, such as poly
(dA:dT), into an RNA-containing 50
-triphosphate moiety, which
can then be sensed by RIG-I leading to IFNβ induction (Ablasser
et al., 2009; Chiu et al., 2009). This provided an explanation for the
previously reported involvement of MAVS in some DNA responses
(Ishii et al., 2006), and certainly accounts for the ability of some
human cell types to respond to poly(dA:dT). However the physiological
role of RNA Pol III in detection of pathogens has remained
controversial, and RNA Pol III could not account for known IFN-I
responses to non-AT-rich dsDNA (Wilkins and Gale, 2010). Thus,
given that the role of DAI is cell type-specific, and that the Pol III-RIGI
pathway only detects AT-rich DNA, clearly additional cytosolic DNA
PRRs remained to be discovered.
The centrality of STING in controlling DNA sensing
In parallel to these early studies identifying putative cytosolic DNA
sensors, a new signaling adaptor protein called STING (also called
MITA, MPYS and ERIS) was discovered in 2008 (Ishikawa and Barber,
2008; Zhong et al., 2008; Jin et al., 2008; Sun et al., 2009). STING was
shown to be essential for IFN-β induction by DNA and also by HSV-1,
and STING knock out mice were subsequently shown to be impaired
in their ability to respond to DNA viruses (Ishikawa et al., 2009).
STING is now known to have a role in vivo in responses to viral and
bacterial pathogens, to self-DNA during autoimmune disease, and in
DNA adjuvancy (Burdette and Vance, 2013). The mechanism whereby
STING directly engages with TBK1 to direct IRF3 activation has also
been determined (Tanaka and Chen, 2012) although how exactly
STING mediates NFκB activation (presumably via IKKβ) is to date still
an open question (Fig. 2) (Abe and Barber, 2014). In addition to its
adaptor function for DNA sensing pathways, STING was shown to
directly recognize and induce a type I IFN response to the bacterial
second messenger cyclic dinucleotide (CDN) diguanylate monophosphate
(c-di-GMP) (Burdette et al., 2011), and more recently to the
structurally related novel mammalian second messenger cGAMP
(described below). The bacterial CDN could be being detected by
STING as a PAMP to sense the presence of a bacterial infection, or
alternatively bacterial CDN stimulation of STING may be utilized as an
immune escape mechanism when induction of type I IFN may benefit
the bacterial pathogen.
Work since 2009 up to the present has now linked STING to
many physiological and pathological responses involving innate and
adaptive immunity, and placed this innate immune adaptor at the
center of both protective and detrimental processes in vivo. For
example, a protective role for STING during cancer immunotherapy
has been revealed in mouse tumor models whereby STING in
dendritic cells is activated by the recognition of tumor cell DNA,
leading to IFN-β induction which renders DCs competent to present
tumor antigens and prime CD8þ T lymphocytes (Klarquist et al.,
2014; Woo et al., 2014; Deng et al., 2014). In contrast, STING has a
detrimental role in driving inflammatory disease when self-DNA
accumulates due to defective or absent DNase activity (Ahn et al.,
2014). Importantly, STING has been directly implicated in human
disease since a new monogenic disorder termed STING-associated
vasculopathy with onset in infancy (SAVI) has been recently
characterized as an autoinflammatory disease caused by gain-offunction
mutations in TMEM173 (the gene encoding STING) (Liu
et al., 2014).
Discovery of multiple putative DNA sensors
Since 2009 at least 10 further proteins have been proposed as
cytosolic DNA sensors: AIM2, IFI16, LRRFIP1, DHX9, DHX36, DDX41,
Ku70, DNA-PK, MRE11, cGAS, STING itself and Rad50 (Figs. 1 and 2).
AIM2 and IFI16 are pyrin and HIN200 domain (PYHIN) proteins that
have been shown to directly bind to DNA and mediate inflammasome
and transcription factor activation respectively (discussed in
detail in the next section).
In 2010 an siRNA screen identified LRRFIP1 as required for IFN-β
gene induction in response to Listeria monocytogenes or dsDNA in
macrophages (Yang et al., 2010). LRRFIP1 was found to not be required
for NF-κB nor IRF3 activation but rather promoted phosphorylation of
the co-activator, β-catenin, leading to recruitment of p300 to the IFNβ
enhanceosome via IRF3 (Wathelet et al., 1998). LRRFIP1 was proposed
to be a DNA sensor since it co-immunoprecipitated with dsDNA,
however direct binding to DNA was not conclusively demonstrated,
and in fact LRRFIP1 also had a role in dsRNA-stimulated IFN-β
induction. Hence, by promoting β-catenin phosphorylation following
nucleic acid detection, LRRFIP1 is capable of indirectly up regulating
IFN-β (Yang et al., 2010).
Many DEAD/H-box helicases, such as RIG-I and MDA5, have
been implicated in innate nucleic acid sensing, but DHX9 and
DHX36 were the first such proteins proposed to sense cytosolic
DNA. These two helicases were identified in plasmacytoid DCs
148 A. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152
(pDCs) by mass spectroscopy as CpG binding proteins, and
surprisingly, shown to signal to altered gene expression via the
TLR adaptor MyD88 (Kim et al., 2010). DHX36 was associated with
IFN-α production and IRF7 nuclear translocation in response to
CpG-A DNA, while DHX9 was found to be important for TNF-α and
IL-6 production and NF-κB activation in response to CpG-B DNA
(Kim et al., 2010). Knockdown of TLR9 in these cells was also found
to almost completely abrogate CpG DNA-dependent responses
(Kim et al., 2010), consistent with the possibility that DHX9 and
DHX36 may be required for the TLR9 pathway in pDCs, rather than
acting as DNA sensors themselves. DHX9 has also been proposed
as a dsRNA sensor in myeloid DCs since knockdown of DHX9 by
shRNA showed DHX9 to be required for the production of type I
IFNs and pro-inflammatory cytokines in response to poly(I:C),
influenza A and reovirus (Zhang et al., 2011). DHX9 was shown to
bind poly(I:C) via its dsRNA-binding domain and signal via MAVS
to trigger activation of NF-κB and IRF3 (Zhang et al., 2011). How
exactly DHX9 might activate MyD88 and MAVS for signaling after
DNA and RNA sensing respectively has not been resolved to date.
In order to identify the potential involvement of other DExD/H
family members in innate immunity, Yong-Jun Liu’s group carried
out a siRNA screen with 59 members of the family. In 2011 they
identified DDX41 as being required for NF-κB and IRF3 activation,
and for cytokine induction, following stimulation of mDCs or human
monocytes with dsDNA or HSV-1 (Zhang et al., 2011). DDX41 was
shown to bind DNA and to also associate with both STING and TBK1,
in keeping with its role as a proposed DNA sensor (Zhang et al.,
2011). More recently, DDX41 was reported to directly bind the
bacterial CDNs, cyclic-di-GMP and cyclic di-AMP (Parvatiyar et al.,
2012). Upon binding to CDNs, DDX41 was shown to induce an IFN-I
response via STING, TBK1 and IRF3 (Parvatiyar et al., 2012). As
DDX41 was shown to bind bacterial CDNs, it may also have a role in
cGAMP sensing (see below) which could contribute to the mounting
of an effective response to DNA virus infection.
Unlike other PRRs such as TLRs and RIG-I, DExD/H-box proteins
implicated in DNA sensing do not have clearly defined signaling
domains that are distinct from their DNA-binding domains. This
makes it difficult to explain how the binding of dsDNA (and of
CDNs in the case of DDX41) would lead to the recruitment of
signaling adapters such as STING, and there have been no followon
studies to date addressing this issue or providing clarity on the
signaling mechanism. Further, in vivo studies demonstrating an
essential role for DExD/H-box proteins in cytosolic dsDNA sensing
have yet to emerge.
Apart from the DExD/H-box family of proteins, proteins known
previously to have a role in responding to DNA damage in the nucleus
have also been implicated as cytosolic DNA sensors. Such proteins
might act independently of their role in the DNA damage response
(DDR) as direct sensors of viral DNA, and/or they may be mobilized to
stimulate innate immune signaling in response to virus-induced
cellular DNA damage in the nucleus (discussed in (Paludan and
Bowie, 2013)). DNA dependent protein kinase (DNA-PK) is a heterotrimeric
protein complex consisting of Ku70, Ku80 and the catalytic
subunit DNA-PKcs. Ku70 is mainly known for its involvement in the
DNA repair process (Walker et al., 2001). However, in 2011 it was also
described as having a role in the cytoplasmic detection of dsDNA of
greater than 500 bp in length, triggering the production of type III IFN
(IFNλ1), rather than IFN-I, via IRF1 and IRF7 activation (Zhang et al.,
2011). A year after identification of Ku70 as a DNA sensor (Zhang et al.,
2011), the DNA-PK complex was identified as a DNA sensor with a
particular role in fibroblasts, where it associated with DNA in the
cytoplasm and signaled via the STING-TBK1-IRF3 axis to IFN-I induction
(Ferguson et al., 2012). DNA-PK colocalised with sites of vaccinia
virus replication and both cells and mice lacking DNA-PKcs showed
attenuated cytokine responses to both DNA and DNA viruses, but
not to RNA or RNA viruses. Interestingly, although crucial for IRF3
activation in response to immunostimulatory DNA or vaccinia virus
infection, DNA-PK was not required for DNA-dependent NF-κB
activation (Ferguson et al., 2012). Further investigations on how
DNA-PK signals via STING for IRF3 activation without NF-κB activation
may shed light on differential mechanisms for DNA-stimulated
transcription factor activation.
Yet another DNA damage sensor protein, Mre11 has also been
shown to mediate STING-dependent cytosolic responses to dsDNA
(but not to DNA virus) (Kondo et al., 2013). Most recently, Rad50,
which is part of a complex together with Mre11 and Nbs1 that senses
and responds to double-strand breaks in nuclear DNA, has also been
implicated in innate immune DNA sensing (Roth, 2014). In that case,
after vaccinia virus infection, Rad50 translocated to the cytosol,
engaged with viral DNA and coupled to a STING-independent signaling
axis involving CARD9 and Bcl10. This led to NF-κB activation, and
in particular induction of pro-IL-1β mRNA. Thus in the context where
DNA viruses cause inflammasome activation, the Rad50-CARD9-
Bcl10-NF-κB pathway contributes to virus-stimulated IL-1β release
(Roth, 2014). The discovery of the role of Rad50 in DNA sensing
suggests that in certain contexts STING-independent DNA response
pathways to gene induction exist.
Apart from its key role as a signaling adaptor in cytosolic DNA
sensing, it has recently been reported that STING binds directly to
DNA both in vitro and in intact cells, possibly leading to direct
activation of the STING signaling pathway (Abe et al., 2013). However,
further work will need to be carried out in order to establish whether
STING can directly sense foreign DNA without the aid of other DNA
sensors in certain contexts, especially since stimulation of HEK293
cells stably expressing STING can induce IFNI in response to CDNs but
not DNA (Burdette et al., 2011).
Mechanisms of innate DNA sensing: lessons from PYHIN
proteins
As well as triggering altered gene induction via transcription factor
activation, cytosolic DNA is also capable of stimulating inflammasome
activity leading to production of the mature active forms of IL-1β and
IL-18. Many inflammasome activators work via stimulation of an
NLRP3-containing inflammasome complex that involves ASC recruitment
and subsequent caspase 1 activation, however cytosolic DNA
was shown to stimulate an NLRP3-independent inflammasome complex.
In 2009, the human PYHIN family member absent in melanoma
2 (AIM2) was shown to detect viral dsDNA in the cytoplasm by direct
binding via the AIM2 HIN200 domain. This facilitated recruitment of
ASC to AIM2, presumably via homotypic PYRIN domain interactions,
and subsequent caspase 1 activation (Roberts et al., 2009; Hornung
et al., 2009; Burckstummer et al., 2009; Fernandes-Alnemri et al.,
2009). Importantly, since there is an ortholog of human AIM2 in the
mouse, the generation of AIM2 knock out mice allowed demonstration
of a role for AIM2 in DNA- and DNA virus-stimulated IL-1β and
IL-18 production in vivo (Fernandes-Alnemri et al., 2010; Rathinam
et al., 2010).
After the discovery of the role of AIM2 in DNA sensing, a second
human PYHIN protein was implicated as a PRR for intracellular
DNA. Interferon-γ-inducible protein 16 (IFI16) was identified as a
novel PRR for DNA based on its association with, and requirement
for IFN-I responses to, a 70 base pair dsDNA derived from the
vaccinia virus genome (Unterholzner et al., 2010). Upon DNA
stimulation (Unterholzner et al., 2010) or infection of cells with
herpes viruses (Horan et al., 2013), IFI16 associated with STING
while siRNA knockdown of IFI16 or its presumed mouse counterpart,
p204 (IFI204), inhibited gene induction induced by DNA or
HSV-1, but not by RNA or RNA virus (Unterholzner et al., 2010).
The structure of the PYHIN proteins is consistent with their
proposed role as cytosolic DNA sensors, as they possess a defined
A. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152 149
signaling (PYRIN) and DNA binding (HIN200) domain. IFI16, p204
and AIM2 form a new family of PRRs, termed the AIM2-like
receptors (ALRs) (Keating et al., 2011), and whether other PYHIN
protein family members also function as ALRs remains to be
determined. Ongoing study of the ALRs has revealed structural and
mechanistic insights into how innate immune DNA sensing operates.
The crystal structure of the AIM2 and IFI16 HIN200 domains bound
to dsDNA provided a rationale for sequence-independent sensing of
DNA, since the contacts between the protein receptors and
the DNA were primarily electrostatic interactions with the
phosphate-sugar DNA backbone (Jin et al., 2012). This is consistent
with the fact that DNA of any sequence, when transfected into
monocytic cells, is capable of eliciting cell death and IFN-I responses.
Thus self-DNA is not discriminated from pathogen DNA based on
nucleotide composition.
Alternatively, self:non-self discrimination might be based on the
aberrant appearance of DNA in the cytosol, and hence the nucleus
would be ‘immune privileged’. However this is also not the case since
IFI16 is mainly nuclear in its expression, and has now been demonstrated
to detect multiple nuclear herpes viruses (reviewed in (Orzalli
and Knipe, 2014)). Thus innate DNA sensing can also initiate from the
nucleus, and not just the cytosol. In fact IFI16 has been shown to
shuttle between the cytosol and nucleus for DNA sensing in both
compartments, depending on the acetylation status of its nuclear
localization sequence (Li et al., 2012). The mechanism by which IFI16
discriminates between pathogen and cellular DNA in the nucleus is
not yet clear. However insights into this puzzle come from recent work
showing that in vitro IFI16 assembles on naked dsDNA strands in a
cooperative manner, which is dependent on homotypic pyrin domain
interactions (Morrone et al., 2014). This leads to the formation of
oligomeric IFI16 foci, reminiscent of the assembly of inflammasomes,
which could be expected to be signaling-competent. Optimal IFI16
polymerization required approximately 150 bp of dsDNA. Although
these observations remain to be shown in intact cells, this work
potentially provides an elegant mechanism for IFI16 to discriminate
between self and non-self-DNA based on ‘measuring’ the length of
naked DNA, since host nuclear DNA should normally be complexed
with histones. Thus the true ligand for DNA sensing by PYHIN proteins
is likely long naked DNA.
IFI16 has also been identified as having a role in the detection of
HIV DNA (Jakobsen et al., 2013; Monroe et al., 2014). In HIV-infected
macrophages, IFI16 bound HIV ssDNA in duplex structure due to
long stem and terminal loop regions that form during the HIV
lentiviral life cycle (Jakobsen et al., 2013). Thus in macrophages IFI16
contributed to the early control of HIV. IFI16 also has a HIV detection
role in abortively infected CD4þ T cells. In that case, the accumulation
of viral DNA replication intermediates caused IFI16-dependent
pyroptosis (Monroe et al., 2014), leading to depletion of T cells. Thus
similar to AIM2, in certain contexts IFI16 can promote inflammasome
activation. This is also true for IFI16 sensing of the herpes
viruses KSHV, HSV-1 and EBV (Kerur et al., 2011; Johnson et al.,
2013; Ansari et al., 2013).
Currently the molecular details as to how in response to viruses
IFI16 signals to the inflammasome in some contexts, and also to
STING for IFN-I induction remain unclear. Activated AIM2, like
NLRP3, has recently been shown to mediate inflammasome signaling
by nucleating pyrin domain filaments of ASC, leading to
clustering of the ASC CARD domains and activation of Caspase-1
(Lu et al., 2014; Cai et al., 2014). Whether IFI16-containing inflammasomes
would function in a similar manner remains to be tested.
Notwithstanding the lack of information as to how IFI16
transmits a downstream signal, the importance of IFI16 in viral
DNA sensing is underscored by the fact that viral immune evasion
strategies have already been identified that target IFI16 function.
Specifically, the HSV-1 protein ICP0 targets IFI16 for proteasomal
degradation in order to suppress IRF3 activation (Orzalli et al.,
2012), while the HCMV protein pUL83 interacts with the pyrin
domain of IFI16 in order to prevent it forming an oligomer, likely
blocking receptor activation (Li et al., 2013).
Discovery of cGAS as a critical DNA sensor acting via STING
In 2013, the discovery of a novel signaling pathway upstream of
STING involving cGAMP synthase (cGAS) represented a significant
advance in our understanding of the signaling mechanisms underpinning
innate DNA sensing. Zhijian Chen’s group identified a factor
synthesized by mammalian cells following stimulation with DNA that
was capable of activating STING (Wu et al., 2013). This factor was
cyclic-GMP-AMP (cGAMP) and it was shown to directly bind STING
and lead to IRF3 activation (Wu et al., 2013; Gao et al., 2013). cGAMP is
very similar in structure to the bacterial CDNs that can bind and
activate STING, suggesting that such CDNs are actually mimicking host
cGAMP as a means of stimulating STING-dependent type I IFNInduction
to benefit the bacteria (Gurtler and Bowie, 2013; Burdette
et al., 2011). Chen’s group also identified the enzyme that generates
cGAMP upon DNA stimulation of cells, from the substrates ATP and
GTP, as cGAS (Sun et al., 2013). This novel cGAS-cGAMP signaling
pathway is reminiscent of the pathway whereby adenylate cyclase
generates the second messenger cAMP from ATP in response to G
protein-coupled receptors. The crystal structures of both free cGAS and
of cGAS bound to DNA have already been solved. This revealed that
cGAS resembles the dsRNA binding protein OAS1 in overall structure,
with modifications in the nucleotide binding region that specify
affinity for dsDNA rather than dsRNA (Gao et al., 2013; Civril et al.,
2013; Kranzusch et al., 2013). In the case of cGAS, enzyme activation
occurs in response to direct binding of DNA to cGAS, which causes a
conformational change allowing access of the nucleotide substrates
into the active site, and subsequent synthesis of cGAMP (reviewed in
(Cai et al., 2014)). Similar to the case for DNA recognition by AIM2 and
IFI16, cGAS contacts dsDNA exclusively via the DNA phosphate backbone,
again leading to nucleotide sequence-independent DNA sensing
(Cai et al., 2014).
Importantly, the role of cGAS in DNA sensing in vivo has been
confirmed since cGAS knock out mice are now available. Cells from
such mice (fibroblasts and BMDMs) were unable to induce IFN and
cytokines in response to DNA or live DNA viruses (vaccinia virus,
HSV-1 and MHV68), while cGAS-/- mice were more susceptible to
lethal infection with HSV-1 or vaccinia virus compared to wild
type mice (Li et al., 2013; Schoggins et al., 2014). In addition,
similar to IFI16, cGAS has been shown to have a role in sensing
HIV-1 since HIV-1-infected cells produced cGAMP, and HIV-1
induced IFN was cGAS-dependent (Gao et al., 2013).
DNA detection by cGAS also provides a mechanism for the
spread of intrinsic innate immunity to DNA viruses from infected
cells into neighboring cells, since Ablasser et al. (2013) recently
showed that cGAMP produced in virally-infected cells can be
transferred into uninfected neighboring cells through gap junctions,
leading to direct activation of STING and induction of IFNs .
Thus antiviral immunity can be rapidly conferred on surrounding
uninfected cells independent of the need for the infected cell to
first produce IFNs.
Conclusion
A vast array of cytosolic DNA sensors have been described in the
past decade, leading to many new insights into when, how and why
innate immune responses to DNA occur. In particular in the case of
IFI16 and cGAS, many studies have now demonstrated key roles for
these PRRs in sensing multiple types of DNA viruses and of retroviruses,
while structural studies with these proteins have confirmed
150 A. Dempsey, A.G. Bowie / Virology 479-480 (2015) 146–152
that innate DNA sensors respond to dsDNA in a sequence-independent
manner. Many questions remain to be answered and given the rapid
rate of progress in this field we can expect further clarification on
many of these in the near future. A key issue is why so many DNA
sensors exist, and what is their relative role in DNA sensing in vivo,
and in different cell types? In particular it is interesting that IFI16 and
cGAS have been shown to both be required for detection of HIV-1 and
HSV-1 so whether these sensors actually cooperate to signal to STING
in a given cell type, and/or operate in distinct cell types is unclear.
Interestingly, Storek et al. (2015) recently showed an essential role for
both IFI16 (p204) and cGAS in the same cell type for IFN I induction
following detection of Francisella novicida DNA during a bacterial
infection, by using the CRISPR/Cas9 system to generate cGAS and
p204 single- and double-knock out cells . In the case of HIV-1 sensing,
the role of IFI16 and cGAS in different cells examined to date correlates
well with the relative expression of these proteins in such cells.
Furthermore, how cytosolic cGAS might be so crucial for sensing
nuclear HSV-1 and for protection against HSV-1 in vivo is unclear, as
are the mechanisms whereby nuclear IFI16 would signal to the STINGTBK1-IRF3
signaling axis in the cytosol.
What is clear to date is that innate immune DNA sensing underpins
many physiological and pathological responses and so further insights
and developments in this area will likely yield new approaches to
treatment of infectious, autoimmune and inflammatory disease.

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