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# IRF3 in viral infections: more than just triggering the interferon response

Marie Bourdon, Caroline Manet, Xavier Montagutelli

► To cite this version:

Marie Bourdon, Caroline Manet, Xavier Montagutelli. IRF3 in viral infections: more than just triggering the interferon response. Genes and Immunity, 2025, 26 (5), pp.419-428. 10.1038/s41435-025-00354-2. pasteur-05221966

HAL Id: pasteur-05221966

https://pasteur.hal.science/pasteur-05221966v1

Submitted on 25 Aug 2025

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1

2 **Title: IRF3 in viral infections: more than just triggering the interferon response**

3

4 Running title: Multiple roles of IRF3 in viral infections

5 Marie Bourdon¹, Caroline Manet², Xavier Montagutelli#¹

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7 **Affiliation:**

8 ¹Institut Pasteur, Université Paris Cité, Mouse Genetics Laboratory, F-75015 Paris, France

9 ²Institut Pasteur, Université Paris Cité, Mouse Genetics, Immunity and Infections Laboratory, F-75015 Paris, France

10 Paris, France

11 # Corresponding author: xavier.montagutelli@pasteur.fr

12

13 **ORCID**

14 Marie Bourdon: 0000-0002-6184-1888

15 Caroline Manet: 0000-0002-7730-8818

16 Xavier Montagutelli: 0000-0002-9372-5398

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**Abstract**

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Interferon regulatory factor 3 (IRF3) is the first transcription factor activating the expression of type I interferons (IFN-I). It is present in the cytoplasm of most cell types under basal conditions and its activation by phosphorylation allows a rapid triggering of the IFN-I pathway in response to viral infection. This activation of IFN-I is amplified by IRF7, the other major IFN-I transcription factor which expression is induced, in most cell types, by the interferon response. However, recent data have shown that the role of IRF3 in viral infection extends beyond the IFN-I pathway. Here, we review the studies investigating the impact of IRF3 deficiencies in infected cells and *in vivo*, in mice and in humans. We discuss the discrepancies between and within studies, between isolated cells and whole organisms. While IRF3 is also involved in other pathological processes, we highlight how the newly discovered functions of IRF3 deepen our understanding of its multiple roles in viral infections, which could stimulate the development of pharmacological manipulation of its biological activities.
## 31 Introduction

Induction of type I interferons (IFN-I) by viruses and other pathogens is a critical step in innate immunity which allows immediate response to aggression by infectious agents. It is triggered by the activation of pattern-recognition receptors (PRRs), such as Toll-like receptors (TLR), and cytosolic sensors such as RIG-I and MDA5 (RLR, RIG-I-like receptors), each of which detects specific classes of pathogen-associated molecular patterns (PAMPs). PRRs can also be activated by the recognition of damage-associated molecular patterns (DAMPs) induced by cell damage in infected cells (1).

Recognition of PAMPs and DAMPs by PRRs triggers a signaling pathway leading to the phosphorylation of interferon regulatory factors (IRFs) by kinases IKKε and TBK1. Activated IRFs form homodimers or heterodimers which translocate to the nucleus and bind cis-regulatory elements on the promoters of IFN-I genes (2). The major IFN-I are IFNα encoded by 13 genes in humans and 14 in mice, and IFNβ which is encoded by the single *Ifnb1* gene in both species (3). Once secreted, IFN-I bind their receptor (IFNAR) which triggers a signaling cascade leading to the expression of interferon stimulated genes (ISGs) with antiviral properties (4, 5). Recent studies have demonstrated that other, non-canonical pathways can induce IFN-I signaling. For instance, PARP9 can bind viral RNA and induce the activation of IRFs through the phosphoinositide 3-kinase (PI3K) and AKT3 pathway (6, 7). Endoplasmic reticulum (ER) stress has also been shown to increase IFN-I induction upon stimulation, either through or independently of TBK1, depending on the stressors (8).

IRFs possess diverse functions contributing to the establishment of innate and adaptive immunity, to the regulation of growth and differentiation of multiple immune cell types and to their potential roles in infectious, inflammatory or autoimmune diseases and cancer, as recently reviewed (9).

Among them, IRF1, IRF3, IRF5, IRF7 and IRF8 are involved in the transcription of IFN-I genes. IRF3
and IRF7 are considered the main transcription activators of IFN-I (9, 10), although IRF1 and IRF8 contribute to this activation, and recent studies suggest that IRF1 could participate in a feedback loop with IRF7 (11). IRF3 was identified in 1995 by Au and colleagues, by homology with the previously identified IRF1 and IRF2 (12). IRF3 is expressed in multiple human tissues (spleen, thymus, prostate, ovaries, etc.) and its expression is not modulated by viral infection nor IFN-I treatment (12).

Over the last two decades, the impact of *Irf3* deficiencies in viral infections has been extensively investigated in humans and in mice (13-16). While *in vitro* studies generally support a critical role for IRF3 in the induction of high levels of IFN-I and in the control of viral replication (14, 17, 18), *in vivo* studies have led to less consistent conclusions, as the susceptibility of *Irf3*-deficient mice was dependent on the virus and on experimental parameters (14). Here, we review these results which shed light on the multiple functions of IRF3 in the host response to viral infections.

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## Structure and functions of IRF3

The *Irf3* gene has a similar structure in mice and humans, with 7 exons encoding the IRF3 protein long of 419 amino acids in mice (427 in humans) which includes five domains necessary for its transcriptional functions (Figure 1A-B) (19, 20). The serine-rich region (SRR) can be phosphorylated by the kinases IKKε and TBK1, resulting in the activation of IRF3. Once activated, IRF3 dimerizes through its IRF association domain (IAD) in the C-terminal region. A nuclear localization signal (NLS) and a nuclear export signal (NES) allow the protein to enter and exit the nucleus, respectively. Lastly, IRF3 binds gene promoters with its N-terminal DNA-binding domain (DBD) forming a helix-turn-helix structure (19, 20).

IRF3 also possesses at least three non-transcriptional activities that were identified in mouse and/or human models. First, the recognition of viral dsRNA by RLR can induce the recruitment of the LUBAC

76
77 protein complex which promotes the polyubiquitination of two lysines in the IAD of IRF3 (Figure 1C, red arrow). Once ubiquitinated, IRF3 interacts with BAX and HSP90, and is recruited to the 78 mitochondria by TOM70, which induces the apoptotic pathway through the release of cytochrome 79 C. This activity, which is independent of IRF3's transcriptional activity, has therefore been named RLR-80 induced IRF3-mediated pathway of apoptosis, or RIPA (21-23). For instance, in monocytes infected 81 with human T cell leukemia virus type 1, intermediates of viral retro-transcription can be detected in 82 the infected cells via the STING pathway and lead to IRF3-mediated apoptosis (24). 83

84 The second non-transcriptional activity of IRF3 was described by Popli and colleagues who 85 investigated the role of IRF3 in NF-κB-mediated inflammation which is a significant contributor to 86 viral pathogenesis. They reported that IRF3 interacts with NF-κB p65 subunit in the cytosol, through 87 a domain between the amino acids 210 and 222, distinct from those needed for RIPA or 88 transcriptional activities (Figure 1C, blue arrow). This interaction prevents NF-κB translocation to the 89 nucleus to induce the expression of proinflammatory genes. This activity is known as repression of 90 IRF3-mediated NF-κB activity, or RIKA (25). Using a conditionally inactivated allele of *Irf3*, the same 91 group recently evaluated the cells responsible for the anti-inflammatory effect of IRF3 in mouse 92 models of respiratory infections with Sendai virus (SeV) and mouse hepatitis virus (MHV) (26). They 93 reported that mice in which *Irf3* was deleted specifically in macrophages showed similar high lung 94 viral load, enhanced inflammatory gene expression, lung inflammation and mortality as mice in which 95 *Irf3* was ubiquitously deleted, indicating that IRF3 expression in macrophage was critical in 96 preventing viral replication, inflammation and pathogenesis. 97

97 Another non-transcriptional activity of IRF3 has been described in mice infected with *Listeria monocytogenes*. Infected macrophages (but not *Irf3*-deficient macrophages) undergo rapid 98 proinflammatory necrotic death which promotes the infection, even in the absence of regulators of 99
100 necrotic death or IRF3 activation. By contrast, the extremely rapid kinetics of IRF3-dependent necrosis
101 prevented viral dissemination upon infection with adenovirus (27).

102 *Irf3* is constitutively expressed in most cell types and the IRF3 protein is very stable and present in
103 the cytoplasm in an inactive form under basal conditions. The activation of its transcriptional activity
104 only requires phosphorylation on serine 386 in humans and on serine 379 in mice, and dimerization,
105 thus enabling prompt transcription of the *Ifnb1* gene encoding the interferon β (IFNβ) protein. IFNβ
106 is excreted in the intercellular space and binds to its receptor present at the surface of the IFNβ-
107 producing and neighbor cells (Figure 2). The activation of the IFNAR receptor triggers the
108 phosphorylation of STAT1/2 proteins which bind the promoters of hundreds of interferon-stimulated
109 genes (ISGs). One of them is *Irf7* which encodes IRF7, another potent activator of IFN-I expression,
110 which amplifies and sustains the IRF3-mediated induction. Unlike IRF3, IRF7 has a short half-life and
111 is constitutively expressed only in a few immune cell types such as plasmacytoid dendritic cells
112 (pDCs), B cells, and monocytes in spleen, thymus, and peripheral blood leukocytes (9, 28). The
113 complementary roles of IRF3 and IRF7 in the induction of IFN-I were first described by Sato and
114 colleagues (16).

115 IRF3 and IRF7 have different affinities for the IFN-I genes. IRF3 binds preferentially to the promoters
116 of *Ifnb1* and *Ifna4* genes, while IRF7 has more affinity for the other IFNα genes (29). IRF3 and IRF7
117 also induce type III IFNs (IFN-III), a family of four IFN-λ in humans and three in mice (30). As with
118 IFN-I, IRF3 has more affinity for the *Ifn1* promoter, while IRF7 binds preferentially to *Ifn2* and *Ifn3*
119 promoters (31, 32).

120 Besides IFN-I genes, IRF3 also directly induces the expression of other genes, notably ISGs, such as
121 *Isg15* (12), *Isg56* (33), *Isg54* (34-36), *Ccl5* (also known as RANTES) (37, 38), and *Cxcl10* (also known
122 as IP-10) (34), by binding to the IFN-stimulated response elements (ISRE) in their promoters. IRF3
also induces the expression of the anti-inflammatory cytokine *Il1rn* (39) and the proapoptotic factors
*Tnfsf10* (also known as TRAIL) and *Pmaip1* (also known as NOXA) (36, 40-42).

125

126 Models for studying the roles of IRF3

The roles of IRF3 in the induction of IFN-I and the response to viral infections have been investigated
in a variety of experimental models, in mice and in humans. In mice, the inactivated allele of *Irf3* (*Irf3*
KO) produced by Sato and colleagues in 2000 (16) has been the model of choice both *in vivo* (13,
14, 16) and *in vitro* on primary cell cultures derived from *Irf3* KO mice, such as bone marrow derived
macrophages (BMDM) and bone marrow dendritic cells (BMDC) (15, 17, 25), while primary cultured
neurons (PCN) have been used for studies on neurotropic viruses (17, 43). Mouse embryonic
fibroblasts (MEF) have also extensively been used as they can be easily derived and cryopreserved
(18, 44-46). Some studies have used plasmacytoid dendritic cells (14, 47) although, since they
constitutively express *Irf7*, they do not allow studying the transcriptional role of IRF3 in the IFN-I
response.

Importantly, this original *Irf3* KO strain was later reported to also carry a null mutation in the adjacent
gene *Bcl2l12*, an anti-apoptotic factor that suppresses DNA damage-induced apoptosis (48).
Consequently, the team that made this original KO strain developed a new floxed *Irf3* mouse strain
to study the consequences of the sole *Irf3* KO mutation (49).

Two other genetically modified mouse *Irf3* alleles were instrumental in the identification of the RIPA
and RIKA activities of IRF3. The *Irf3-S1* mutant has functional RIPA and RIKA but no transcriptional
activity, while the *Irf3-M1* mutant has RIKA activity but no RIPA nor transcriptional activity (21, 25).
144 Human studies have taken advantage of polymorphisms in the *Irf3* gene identified from the whole
145 exome sequencing of patients with severe illness following viral infection (50, 51), or sequences of
146 candidate genes including *Irf3* (52, 53). These alleles were tested for association with susceptibility
147 to viral infections (54-56). *In vitro* studies have used complete *Irf3* ablation using CRISPR/Cas
148 techniques, or reduced IRF3 cytoplasmic concentrations through knockdown (KD) techniques such
149 as siRNA or shRNA (25, 45).

150

151 **IRF3 deficiencies lead to decreased IFN-I induction and increased viral**
152 **replication in vitro**

153 *In vitro* studies investigating the role of IRF3 in viral infections have used different cell types, viruses,
154 multiplicity of infection (MOI) and time points, all of which must be considered when comparing the
155 results. Despite this heterogeneity, these studies have generally concluded on the importance of IRF3
156 in the induction of IFN-I and the control of viral replication in human and mouse cellular models.
157 Their principal findings are summarized in Table 1, while a few examples are discussed below.

158 For instance, Wood and colleagues studied mouse gammaherpes virus (MGHV) infection in wild-
159 type (WT) and *Irf3*-deficient BMDM. They reported reduced expression of *Ifnb1* and IFNα genes in
160 cells at 4 hours post-infection (hpi) and reduced levels of IFN-I in the supernatant at 3 and 4 hpi in
161 *Irf3*-deficient BMDM compared with control macrophages. Moreover, at 8 hpi, which corresponds to
162 the peak expression of *Rsad2* and *Mx1* in WT BMDM, these two ISGs were not expressed in *Irf3*-
163 deficient macrophages (57).

164 Moore and colleagues investigated the consequence of an *Irf3* KO on Theiler's encephalomyelitis
165 virus (TMEV) infection in mouse primary macrophages (58). They showed that viral loads in cell
166 lysates were higher in *Irf3* KO macrophages than in WT macrophages from 7 to 24 hpi, suggesting
that *Irf3* was required to control TMEV genome replication. At 3 hpi, expression of *Ifnb1* and *Il6* was lower in *Irf3*-deficient macrophages compared with WT macrophages, but higher at 24 hpi. The authors hypothesized that the higher replication rate in *Irf3* KO macrophages led to delayed, IRF3-independent induction of IFNβ and IL6, for instance through TMEV-induced expression of *Irf1*. The late increased induction of IL6 could also be explained by the absence of the RIKA non-transcriptional activity of *Irf3* in *Irf3*-deficient cells (25).

The role of RIKA has been evaluated in two models of infection in BMDM derived from *Irf3* KO or *Irf3*-M1 mutant mice (21, 25). In the case of MHV, the RIKA domain was sufficient to inhibit viral replication despite the absence of *Ifnb1* induction. Likewise, *Irf3*-M1 BMDM showed partial reduction of SeV RNA compared to *Irf3*-KO BMDM, with a complete repression of *Cxc15*.

Notably, *Irf3*-deficient and *Irf3 Bcl2l12* double-deficient MEFs showed the same reduced induction of IFN-I after infection with encephalomyocarditis virus (EMCV), herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV), showing that *Bcl2l12* loss of function did not modify the effect of *Irf3* KO in these two models (14, 16, 49).

The role of *Irf3* has also been assessed by using siRNA to reduce the amount of mRNA, as illustrated in a study of influenza A virus (IAV) infection in primary human macrophages. *Irf3* KD cells showed lower expression of *Ifnb1* and *Ifn1* at 3 and 6 hpi. As expected from the reduced IRF3-associated RIKA activity, the expression of other cytokines, such as *Cxcl10*, *Ccl2*, *Ccl3* and *Ccl5* was also decreased in *Irf3* KD cells (59). However, *Tnfa* expression was also decreased despite reduced RIKA activity, possibly through its reduced binding to and activation of the *Tnfa* promoter (60).

Cell type-specific consequences of IRF3 deficiency were reported by Daffis and colleagues in a study on WNV infection in mouse BMDM and PCNs (17). Upon infection, *Irf3*-deficient PCN showed reduced IFN-I gene expression and concentrations of IFNα and IFNβ at 24 and 48 hpi in the
supernatants compared with WT cells, associated with a moderate increase in viral load at 48 hpi.

Irf7 induction was also delayed and decreased in Irf3-deficient PCN, which may be the cause of the

low levels of IFN-I. Conversely, in BMDM, while the viral titer was higher in Irf3-deficient cells than in

WT cells at 24 to72 hpi, Irf3-deficient BMDM had earlier and higher expression of IFN-I genes than

WT BMDM. This induction was possibly driven by the increased expression of Irf7 in Irf3-deficient

BMDM, in response to increased viral infection. While it is possible that the time points studied

missed the early IRF3-dependent IFN-I induction which was observed before 24h in other studies on

Irf3 KO BMDM (25, 49, 57), these results demonstrate that the consequences of IRF3 deficiency on

the control of viral infection in vitro may be cell-type specific, in particular regarding the

compensatory role of Irf7.

Variable and multiple impacts of IRF3 deficiencies in vivo

As with *in vitro* experiments, mouse *in vivo* studies have been performed under diverse conditions,

in particular using different viruses, doses and routes of infection (most often intraperitoneal,

subcutaneous, intravenous and intranasal, occasionally intracranial or corneal). The mechanisms

driving the progression of *in vivo* infections are more complex than in *in vitro* models as they involve

interactions between multiple cell types and tissues. Importantly, as for *in vitro* experiments, studies

comparing *Irf3* WT and *Irf3* KO mice were performed on the C57BL/6J background and were

therefore not affected by the influence of other genetic susceptibility factors.

A compilation of the *in vivo* results published using *Irf3* KO mice is presented in Table 2, which shows
less consistency in the consequences of IRF3 deficiency across mouse models than in *in vitro* studies.

In some experiments, *Irf3* KO mice showed higher viral load compared with WT mice in the tissues

tested, while others did not detect significant differences. When measured, IFN-I levels were often

found to be decreased but comparisons between studies are rarely possible due to different
experimental conditions or different time points. As observed *in vitro*, *Irf3*-deficient and *Irf3 Bcl2l12* double-deficient mice showed the same mortality following EMCV infection, indicating that *Bcl2l12* loss of function did not modify the effect of *Irf3* KO in these models (14, 16, 49).

Interestingly, a few studies have examined both *in vitro* and *in vivo* phenotypes and found consistent effects of IRF3 deficiency (see Table 1 and Table 2). For instance, Yanai and colleagues have reported that HSV-1, VSV, and EMCV infection of *Irf3* KO MEF, BMDC and BMDM resulted in lower *Ifnb1* expression than in WT cells. After intravenous infection with EMCV, *Irf3*-deficient mice exhibited a higher mortality rate than WT mice and lower levels of IFNβ in the serum (49).

Similarly, HSV-1 infection of *Irf3*-deficient BMDC led to higher viral titer and lower levels of IFNβ in the supernatant than in WT BMDC (15). *Irf3* KO mice infected with HSV-1 either by the corneal and intracranial route showed a higher mortality rate than WT mice and higher viral titers in the brain. After intracranial infection, *Irf3* KO mice also presented increased levels of cytokines in the brain, such as TNFα, IL10 and CCL5 (64).

In other reports, the susceptibility of *Irf3*-deficient cells was not associated with an increased susceptibility *in vivo*. Upon HSV-1 infection, *Irf3* KO MEF showed a decreased induction of IFN-I, especially IFNβ, compared with WT MEF. However, both *Irf3* KO mice and WT mice survived infection and exhibited the same level of serum IFNα (14). Similarly, infection of *Irf3* KO MEF with Chikungunya virus (CHIKV) induced lower expression of *Ifnb1* compared with WT MEF, while *Irf3* KO and WT mice had the same viral load in the blood and equally survived the infection (18).

These studies, along with others (14, 16, 17, 49, 58), have reported increased susceptibility in *Irf3* KO mice mostly in cases where WT mice displayed clinical signs of illness and mortality. On the other hand, studies that found no effect of IRF3 deficiency on the outcome of viral infection were generally
performed under conditions where infection had no effect on WT mice (14, 18, 62, 65-68) as a
consequence of the low intrinsic virus pathogenicity or of the route and dose of infection.

Differences between studies can be caused by different routes of infection, as exemplified with HSV-
1. Honda and colleagues infected mice intravenously and observed 100% survival and the same levels
of serum IFNα in WT and *Irf3* KO mice (14). On the other hand, Canivet and colleagues infected mice
intranasally and reported a 10% survival rate in *Irf3* KO mice while 70% of WT mice survived infection.
*Irf3* KO mice also showed increased body weight loss and viral titers as well as higher concentrations
of inflammatory cytokines in the brain compared with WT mice (69). Corneal and intracranial HSV-1
infection also led to higher mortality rates in *Irf3*-deficient mice than in WT mice (15).

*In vivo* studies in mice have also allowed investigating the impact of IRF3 deficiency on the kinetics
of IFN-I response over several days. For example, in their above-mentioned study, Canivet and
colleagues reported that, after intranasal HSV-1 infection, levels of IFNβ in brain homogenates were
slightly lower at 3 days post-infection (dpi) but higher at 5 dpi in *Irf3* KO mice compared with WT
mice. The authors suggested the implication of alternative IFN induction pathways to explain the
increased IFN-I production at 5 dpi (69).

Notably, the role of non-transcriptional activities of IRF3 in viral infections has been investigated and
characterized *in vivo*. Mice carrying a mutated allele preserving functional RIPA and RIKA but not
IRF3 transcriptional activity were more susceptible to SeV than *Irf3* WT mice, but less than *Irf3* KO
mice. Moreover, *Irf3*-deficient mice were more susceptible to SeV infection than *Ifnar1*-deficient
mice, which are unable to respond to IFN-I signaling. These results show that the non-transcriptional
activities of IRF3 provide complementary antiviral pathways to the IFN-I induction and are required
for a full IRF3-mediated protection against viral infection (21).
258 IRF3 deficiency did not always result in increased mortality rate, viral loads, or decreased IFN-I titers,
259 depending on the virus studied or on the experimental conditions. However, other parameters may
260 be modified, in particular the inflammatory response to infection which was found to be increased
261 in several studies (Table 2). For instance, after infection with Sindbis virus (SINV), *Irf3*-deficient mice
262 showed higher expression of *Ccl2*, *Il1b*, *Tnfa* and *Il10* at 5 dpi in the brain and/or the spinal cord than
263 WT mice. Moreover, the histological inflammation score was higher in *Irf3* KO than in WT mice (70).
264 Likewise, *Irf3*-deficient mice showed higher levels of IL1α, IL1β, IL6, IL12p40, IL12p70 and IFNγ at 5
dpi after HSV-1 infection than WT mice (69). Whether these effects are due to the absence of RIKA
265 activity or indirectly caused by the lack of IRF3-dependent IFN-I expression was not established but
266 could be investigated using an *Irf3* allele in which the RIKA domain has been altered.
267

268 Finally, a few *in vivo* studies have revealed the role of IRF3 deficiency on the adaptative immune
269 responses. Indeed, IRF3-mediated IFN-I signaling has been shown to mediate T cell differentiation
270 in the context of infections (71). Under basal conditions, the composition of lymphocyte populations
271 is similar between *Irf3* KO and WT mice (16). However, after TMEV or IAV infection, *Irf3*-deficient mice
272 showed reduced proportion of circulating CD8 T cells expressing granzyme B, an enzyme involved
273 in the cytotoxicity of CD8 T cells, and impaired development of memory T cell population (72). After
274 SINV infection, *Irf3*-deficient mice showed increased infiltration of CD4 T cells and CD19 B cells in
275 the brain (70). These two examples underscore the importance of investigating broadly the immune
276 response to infection to get a complete overview of the impact of IRF3 deficiency.
277

278 Human *in vivo* investigations were based on patients with severe viral disease and found to carry
279 mutations in the *IRF3* gene. For example, two dominant mutations were found in patients with herpes
280 simplex encephalitis (HSE). The c.854G>A (R285Q) mutation causes IRF3 to fail to be phosphorylated,
to undergo dimerization, and therefore to activate transcription, resulting in impaired IFN-I
281 responses in peripheral blood mononuclear cells (PBMC) infected with HSV-1 (50). The c.829G>A (A277T) was also found to inhibit induction of *IFNB1* and *CXCL10* expression in HSV1-infected PBMC
282
283 (52). A non-coding *IRF3* variant (rs2304204; -925A>G) was associated with liver cirrhosis induced by
284 hepatitis C virus in Egyptian patients, with the heterozygous A/G genotype being protective (55). This
285 polymorphism was not found to be associated with chronic hepatitis B virus infection in a cohort of
286 Chinese patients (56).

287 A dominant c.1576C>T variant in the 3'UTR of *IRF3* was found by whole exome sequencing in a
288 patient with severe IAV disease. PBMC showed normal mRNA expression but reduced amounts of
289 IRF3 protein, suggesting that the variant could affect *IRF3* mRNA translation into protein. Upon
290 infection with IAV, PBMC exhibited impaired and almost abolished induction of IFN-I and IFN-λ
291 compared with controls (51). Similarly, two dominant mutations (p.Glu49del/WT and
292 p.Asn146Lys/WT) were found in patients with severe COVID19, which importance was confirmed by
293 decreased *IFNB1* induction in transfected HEK293T cells (53). Lastly, in the case of WNV infection, the
294 alternative C allele for the rs2304207 SNP in intron 2 of *IRF3* was overrepresented in symptomatic
295 cases compared with asymptomatic cases (54). In conclusion, unlike mice in which *Irf3* variants were
296 shown to have a variable impact depending on the virus tested, human studies on *IRF3* have been
297 restricted to cases where deficiencies result in severe disease following viral infection.

298

299 **Complementary roles of IRF3 and IRF7**

300 IRF3 and IRF7 are the two main transcriptional activators of IFN-I. While the main role of the
301 constitutively expressed IRF3 is to initiate very rapidly *Ifnb1* transcription, IRF7 which is expressed as
302 an ISG in most cell types sustains and amplifies IFN-I expression. However, in specific cell types such
303 as pDCs, it can also be activated by TLR/RLR pathways and induce IFN-I gene expression (9, 16).
Because of these functional differences, *Irf3* and *Irf7* deficiencies have different consequences *in vitro* and *in vivo*. This was initially demonstrated in 2005 by Honda and colleagues who compared *Irf3* and *Irf7* deficiencies in MEF. Infection with HSV-1, vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) resulted in a more severe reduction of IFN-I gene expression in *Irf7* KO than in *Irf3* KO MEF. Induction of IFN-I gene expression was also reduced upon infection in *Irf7* KO pDCs compared with WT pDCs, but not in *Irf3* KO pDCs. This result is consistent with the constitutive expression of *Irf7* in this cell type which drives IFN-I expression without the need for IRF3-driven induction. *In vivo*, *Irf7*-deficient mice were more susceptible to HSV-1 and EMCV infection than *Irf3*-deficient mice (14).

A number of *in vivo* studies have compared the consequences of IRF3 and IRF7 deficiencies on viral infections (14, 18, 73, 76). Mice with an *Irf7* KO mutation were generally reported to show more severe infection (viral load, signs of disease, mortality) than those with *Irf3* KO (14, 68, 70, 73). Studies on *Irf3 Irf7* double KO mice have most often reported that IRF3 deficiency potentiates the susceptibility observed in *Irf7* single KO mice. For instance, after infection with SINV, *Irf7*-deficient mice showed a mortality rate of 100%, while all *Irf3*-deficient and WT mice survived. All *Irf3 Irf7* double-deficient mice died, like *Irf7* KO mice, but more rapidly (70). Likewise, after IAV infection, all WT mice survived while the mortality rate was 40% in *Irf3* KO mice, 60% in *Irf7* KO mice, and 80% in *Irf3 Irf7* double-deficient mice (76). In the case of two studies on CHIKV and Oropouche virus (OROV) (66, 68), only *Irf3 Irf7* double-deficient died from the infection, while WT, *Irf3* KO and *Irf7* KO mice survived. The effect of *Irf3 Irf7* double deficiency has also been reported on viral load in tissues. For instance, after DENV infection, *Irf3 Irf7* double KO mice have increased viral loads in the serum, spleen, liver and kidney, compared with single KO and WT individuals (65).
326 Other studies have highlighted more complementary roles for IRF3 and IRF7 against viral infection.

327 For example, after IAV infection, lung viral titer was increased in *Irf3* KO mice while it was similar in

328 *Irf7* KO and WT mice (76). In bronchoalveolar lavages, IFNβ levels were significantly lower in *Irf3* KO

329 than in WT mice at 1 and 2 dpi, while they were reduced only at 1 dpi in *Irf7* KO mice. By contrast,

330 IFNα levels were much lower in *Irf7* KO than in WT mice at 1 and 2 dpi, while they were lower in *Irf3*

331 KO than in WT mice at 1 dpi but similar at 2 dpi. These results are consistent with the known

332 preferential affinity of IRF3 and IRF7 for the promoters of the *Ifnb1* and *Ifna* genes, respectively (29).

333 Interestingly, IFN-λ levels in bronchoalveolar lavages were lower in *Irf7* KO than in WT mice at 1 and

334 2 dpi, but were four-fold higher in *Irf3* KO than in WT mice at 2 dpi, suggesting a compensatory

335 mechanism in the respiratory tract of *Irf3*-deficient mice (76).

336 *Irf3* and *Irf7* deficiencies may also have different effects depending on the organ tested or on mouse

337 age. Upon infection with murine norovirus (MNV), viral titer was increased in the lung, spleen and

338 lymph node of *Irf3* KO and *Irf7* KO mice, while only *Irf3*-deficient mice showed increased viral titer in

339 the ileum compared to WT mice (73). In the case of CHIKV infection, adult mice lacking either *Irf3* or

340 *Irf7* survived infection as well as WT mice, while 100% of 9-day-old *Irf3* KO and *Irf7* KO mice

341 succumbed, compared with 50% in WT mice (18).

342

343 **Conclusions and perspectives**

344 IRF3 is a major component of the innate immune response, allowing an immediate activation of

345 antiviral effector proteins. After its discovery in 1995, it was extensively studied for its transcriptional

346 activity and has often been considered in the context of the other main IFN-I transcription factor,

347 IRF7. However, IRF3 possesses other non-transcriptional activities, RIPA, RIKA and proinflammatory

348 necrotic death, extending its range of action against viral infections beyond interferon signaling. The
349 recent description of these additional properties of IRF3, and of the presence of a loss-of-function mutation in the *Bcl2l12* gene in the *Irf3* KO strain used in most studies, should encourage refining the evaluation of the impact of IRF3 in the response to viral infections.
350
351

352 In this review, we have highlighted the complementarity of information provided by studies on the effect of IRF3 deficiency in cellular models and in whole organisms. While *in vitro* studies have largely supported the importance of *Irf3* in triggering the expression of IFN-I and in controlling viral replication, *in vivo* experiments have led to more heterogeneous conclusions, for multiple reasons including the virus, the mouse characteristics, the conditions of infection, the time points or the parameters measured. Several studies reported altered IFN-I signaling and increased viral replication in *Irf3*-deficient cellular models, while the susceptibility of *Irf3* KO mice was not increased compared to WT mice (14, 18, 62). Moreover, we presented examples where the impact of IRF3 deficiency was variable between organs and cell types. These observations emphasize the limitations of every *in vivo* study in capturing a comprehensive understanding of the mechanisms impacted by the loss of IRF3 and illustrate the intrinsic restrictions of *in vitro* experiments involving a single cell type to provide a reliable proxy of the pathophysiology of viral infections. This is of particular concern regarding human studies based on isolated patients with severe signs of illness. The identification of a mutation in the *IRF3* gene, with a confirmed defect of IFN-I expression in a cell type isolated from the patient, is suggestive but does not, by itself, prove that this mutation is responsible for the high susceptibility of the patient to the infection. A compelling example is provided by our recent identification of an *Irf3* loss of function mutation in the CC071 mouse strain that exhibits high susceptibility to ZIKV and other viruses (62). MEF from CC071 and *Irf3* KO mice were equally unable to control viral replication and showed the same delay in *Ifnb1* transcription compared with WT MEF. However, *Irf3*-deficient mice exhibited the same peak viral load as WT mice, without clinical signs of illness. These results demonstrate that other genes are required to explain the high susceptibility of
373 CC071 mice. The identification of these genes may enhance our understanding of the host response
374 to viral infection, in synergy with or independently from *Irf3*.

375 The accumulation of genomic data in humans will undoubtedly reveal a myriad of polymorphisms in
376 the *Irf3* gene. In humans as in mice, although a mutation in *IRF3* is an attractive candidate, the role
377 of other genes cannot be excluded. The identification of these genes may enhance our
378 understanding of the host response to viral infection, in synergy with or independently from *Irf3*.
379 However, CRISPR-based engineering technologies can be used to study the impact of such mutations
380 on the protein function in cellular models. By studying different domains, such methods will be
381 instrumental in better understanding their role, discovering new functions, and possibly identifying
382 new therapeutic potentials.

383 The majority of the studies reported in this review have been conducted using mouse models or cells
384 cultured from them. This calls into question the validity of these data for humans. While the general
385 organization and domains of the *Irf3* gene (including DNA-binding domain and IAD domain) are
386 conserved and its transcriptional, RIPA and RIKA activities have been confirmed in human cells, there
387 are subtle but important differences in how phosphorylation activates IRF3 in each species (79).
388 Moreover, properties and biological implications that were established from genetically engineered
389 mouse mutants cannot be easily tested in humans.

390 The influence of sex on IFN-I responses has been recognized for a long time, with females building
391 stronger innate and adaptive immune responses than males, resulting in faster clearance of
392 pathogens (80-82). *Tlr7*, which recognizes single-stranded RNAs and is located on the X
393 chromosome, was shown to escape X-inactivation and is therefore overexpressed in females (83).
394 Moreover, *Irf5* shows higher basal expression in female pDCs under the control of estrogen receptor
395 1 (84), providing a mechanism for higher IFNα expression upon stimulation with TLR7 agonists.
396 Notably, sex was never mentioned in the *in vitro* studies reviewed here, while two *in vivo* infection 397 studies were done on female mice only (72, 76), one on males only (69) and two studies reported no 398 differences between males and females (62, 64). This analysis shows that the effect of sex is still 399 insufficiently taken into account in many studies, particularly *in vitro*. Often, the sex of the animals or 400 cells is not mentioned. Considering the general tendency of females to be less susceptible to 401 infectious diseases, partly due to a more efficient interferon pathway, it is recommended to identify 402 and report the sex of the biological resources used and to compare responses in both sexes 403 whenever possible. 404

404 Due to IRF3's key role in antiviral immunity, this factor is antagonized by several viruses. Its activity 405 can be inhibited by viral molecules by targeting different steps of its activation pathway, for instance, 406 the dimerization or the nuclear translocation, or by inducing the degradation of the protein (20). 407 Other example are herpes simplex virus 1 (HSV-1) kinase US3 which was shown to 408 hyperphosphorylate IRF3 preventing its dimerization (85), and varicella-zoster virus (VZV) ubiquitin 409 ligase ORF61 which can induce the degradation of IRF3 via the ubiquitin-proteasome pathway (86). 410

410 Finally, the knowledge accumulated on the multiple functions of IRF3 in a variety of immune 411 mechanisms and diseases (9) has led to consider it as a potential drug target. Indeed, IRF3 agonists 412 have been demonstrated to robustly induce antiviral genes, reduce viral burden and improve survival 413 in preclinical models. For instance, the IRF3 agonist KIN1148 improved the survival of mice infected 414 with enterovirus A71. Animals treated with the agonist showed increased IFN-I response and 415 decreased inflammation (87). IRF3 agonists were also tested as vaccine adjuvants. The administration 416 of KIN1148 as an adjuvant to the IAV vaccine increased the antibody responses of vaccinated mice 417 and their survival after viral challenge (88). The latest discoveries on IRF3 non-transcriptional activities 418 also offer new therapeutic potential. Doxorubicin and pyrvinium pamoate were shown to induce RIPA 419
419 in cellular models, inhibiting the replication of multiple viruses (89). These data demonstrate that
420 deepened understanding of IRF3 properties may lead to new pharmacological approaches to
421 complement current therapeutic interventions fighting viral infections and, more broadly, the many
422 immunological and inflammatory disorders which course is influenced by IRF3.

423

424 Acknowledgements

425 This work was supported by grants from the Agence Nationale de la Recherche (ANR) NeuroZika
426 (ANR-20-CE16-0017), from the French Government's Investissement d'Avenir program, Laboratoire
427 d'Excellence: IBEID (Integrative Biology of Emerging Infectious Diseases, ANR-10-LABX-62-IBEID)
428 and from MSDAVENIR (grant INTRANZIGEANT). C.M. and M.B. were supported by doctoral
429 fellowships from grant ANR-10-LABX-62-IBEID.

430

431 Author Contributions

432 M.B. and X.M. conceived and wrote the paper with suggestions and revisions from C.M.

433

434 Competing Interests

435 The authors declare that they have no competing interests.

436
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664 88. Probst P, Grigg JB, Wang M, Munoz E, Loo YM, Ireton RC, et al. A small-molecule IRF3 agonist

665 functions as an influenza vaccine adjuvant by modulating the antiviral immune response. Vaccine.

666 2017;35(15):1964-71.

667 89. Glanz A, Chawla K, Fabry S, Subramanian G, Garcia J, Jay B, et al. High Throughput Screening

668 of FDA-Approved Drug Library Reveals the Compounds that Promote IRF3-Mediated Pro-Apoptotic

669 Pathway Inhibit Virus Replication. Viruses-Basel. 2020;12(4):442.
<table><caption>Table 1: Consequences of *Irf3* deficiencies in cellular models of viral infection</caption><thead><tr><th>Family</th><th>Virus</th><th>Species</th><th>Cell</th><th>Allele</th><th>IFN</th><th>Viral load</th><th>Other</th><th>Reference</th></tr></thead><tbody><tr><td>Adenoviridae</td><td>AdV</td><td>Human</td><td>A549</td><td>Irf3 KO<sup>3</sup></td><td>=</td><td>=</td><td>ND</td><td>(61)</td></tr><tr><td rowspan="2">Coronaviridae</td><td rowspan="2">MHV</td><td>Mouse</td><td>BMDM</td><td>Irf3 KO<sup>1</sup></td><td>ND</td><td>↑</td><td>↑ (inf)</td><td>(25)</td></tr><tr><td>Mouse</td><td>BMDM</td><td>Irf3-M1<sup>4</sup></td><td>ND</td><td>↓</td><td>ND</td><td>(25)</td></tr><tr><td rowspan="4">Flaviviridae</td><td rowspan="2">WNV</td><td>Mouse</td><td>MEFs</td><td>Irf3 KO<sup>1</sup></td><td>NA</td><td>↑</td><td>NA</td><td>(44)</td></tr><tr><td>Mouse</td><td>BMDM</td><td>Irf3 KO<sup>1</sup></td><td>↑ (IFNα &amp; β)</td><td>↑</td><td>↑ (Irf7)</td><td>(17)</td></tr><tr><td rowspan="2">ZIKV</td><td>Mouse</td><td>PCN</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNα &amp; β)</td><td>↑</td><td>↓ (Irf7)</td><td>(17)</td></tr><tr><td>Mouse</td><td>MEF</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>ND</td><td>(62)</td></tr><tr><td rowspan="10">Herpesviridae</td><td rowspan="10">HSV-1</td><td>Mouse</td><td>MEF</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>ND</td><td>ND</td><td>(14)</td></tr><tr><td>Mouse</td><td>pDC</td><td>Irf3 KO<sup>1</sup></td><td>= (IFNα)</td><td>ND</td><td>ND</td><td>(14)</td></tr><tr><td>Mouse</td><td>MEF</td><td>siRNA</td><td>↓ (IFNβ)</td><td>ND</td><td>ND</td><td>(45)</td></tr><tr><td>Mouse</td><td>BMDC</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>ND</td><td>(15)</td></tr><tr><td>Mouse</td><td>BMDM</td><td>Irf3 KO<sup>1</sup></td><td>ND</td><td>↑</td><td>ND</td><td>(15)</td></tr><tr><td>Mouse</td><td>BMDC</td><td>Irf3 KO<sup>1</sup></td><td>ND</td><td>↑</td><td>ND</td><td>(63)</td></tr><tr><td>BMDC,</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BMDM,</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MEF</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MGHV</td><td>Mouse</td><td>BMDM</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>↓ (ISG)</td><td>(57)</td></tr><tr><td rowspan="2">Orthomyxoviridae</td><td rowspan="2">IAV</td><td>Human</td><td>PrM</td><td>siRNA</td><td>↓ (IFNβ)</td><td>ND</td><td>↓ (ISG, inf)</td><td>(59)</td></tr><tr><td>Mouse</td><td>BMDM</td><td>Irf3 KO<sup>1</sup></td><td>ND</td><td>↑</td><td>↑ (inf)</td><td>(25)</td></tr><tr><td rowspan="4">Paramyxoviridae</td><td rowspan="2">NDV</td><td>Mouse</td><td>MEF</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNα &amp; β)</td><td>ND</td><td>ND</td><td>(16)</td></tr><tr><td>Human</td><td>HT1080</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>↑ (inf)</td><td>(25)</td></tr><tr><td rowspan="2">SeV</td><td>Mouse</td><td>BMDM</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>↑ (inf)</td><td>(25)</td></tr><tr><td>Mouse</td><td>BMDM</td><td>Irf3-M1<sup>4</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>= (inf)</td><td>(25)</td></tr><tr><td>Phenuiviridae</td><td>RVFV</td><td>Human</td><td>A549</td><td>Irf3 KO<sup>3</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>↓ (Irf7)</td><td>(61)</td></tr><tr><td rowspan="4">Picornaviridae</td><td rowspan="2">EMCV</td><td>BMDC,</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>BMDM,</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MEF</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Mouse</td><td>MEF</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>ND</td><td>ND</td><td>(49)</td></tr><tr><td>TMEV</td><td>Mouse</td><td>PM</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>↑</td><td>↓ (Irf7), ↑ (inf)</td><td>(58)</td></tr><tr><td>Poxviridae</td><td>Myxoma</td><td>Mouse</td><td>pDC</td><td>Irf3 KO<sup>1</sup></td><td>= (IFNα &amp; β)</td><td>ND</td><td>ND</td><td>(47)</td></tr><tr><td>Rhabdoviridae</td><td>VSV</td><td>Mouse</td><td>MEF</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>ND</td><td>ND</td><td>(14)</td></tr></tbody></table>
<table><tbody><tr><td></td><td></td><td>BMDC, BMDM, MEF</td><td>Irf3 KO<sup>2</sup></td><td>↓ (IFNβ)</td><td>ND</td><td>ND</td><td>(49)</td></tr><tr><td></td><td>Mouse</td><td>pDC</td><td>Irf3 KO<sup>1</sup></td><td>= (IFNα)</td><td>ND</td><td>ND</td><td>(14)</td></tr><tr><td></td><td>Mouse</td><td>MEF</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>ND</td><td>ND</td><td>(18)</td></tr><tr><td rowspan="2">Togaviridae</td><td>CHIKV</td><td>Mouse</td><td>PCN</td><td>Irf3 KO<sup>1</sup></td><td>↓ (IFNβ)</td><td>=</td><td>↓ (cell viability)</td><td>(43)</td></tr><tr><td>WEEV</td><td>Human</td><td>BE(2)-C</td><td>dn</td><td>↓ (IFNβ)</td><td>↑</td><td>↓ (cell viability)</td><td>(43)</td></tr></tbody></table>

671

672

673

674

675

676

677

678

679

680

681

682

683

A549: adenocarcinomic human alveolar basal epithelial cell line A549; AdV: adenovirus; BE(2)-C: human neuroblastoma cells; BMDC: bone marrow dendritic cells; BMDM: bone marrow derived macrophages; CHIKV: Chikungunya virus; dn: dominant negative allele; IAV: influenza A virus; inf: inflammation; EMCV: encephalomyocarditis virus; HSV-1: herpes simplex virus 1; MHV: murine hepatitis virus; MEF: mouse embryonic fibroblasts; MGHV: mouse gammaherpes virus; ND: not determined; NDV: Newcastle disease virus; PCN: primary cultured neurons; pDC: plasmacytoid dendritic cells; PM: peritoneal macrophages; PrM: primary macrophages; RVFV: Rift Valley fever virus; SeV: Sendai virus; TMEV: Theiler's murine encephalomyelitis virus; VSV: vesicular stomatitis virus; WEEV: Western equine encephalitis virus; WNV: West Nile virus; ZIKV: Zika virus; 1: *Irf3*<sup>tm1Ttg</sup>, 2: *Irf3*<sup>tm2.1Ttg</sup>; 3: CRISPR/Cas9 KO in vitro; 4: *Irf3* allele described in (21); ↑: higher in *Irf3* KO than in WT cells; ↓: lower in *Irf3* KO than in WT cells; =: not significantly different between *Irf3* KO and WT cells.
<table><caption>684 Table 2: Consequences of *Irf3* KO *in vivo* in mouse models of viral infection</caption><thead><tr><th>Family</th><th>Virus</th><th>Route</th><th>IFN</th><th>Inflammation</th><th>Viral load</th><th>Clinical signs</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="3">Calciviridae</td><td>MNV</td><td>O</td><td>ND</td><td>ND</td><td>↑ (liver, LN, ileum, spleen)</td><td>ND</td><td>(73)</td></tr><tr><td>DENV</td><td>IV</td><td>= (serum)</td><td>ND</td><td>= (kidney, liver, spleen, serum)</td><td>= (S)</td><td>(65)</td></tr><tr><td>FP</td><td>= (serum)</td><td>ND</td><td>ND</td><td>↑ (brain, kidney, LN, serum, spleen, SC)</td><td>ND</td><td>(74)</td></tr><tr><td rowspan="6">Flaviviridae</td><td>WNV</td><td>FP</td><td>↑ (serum) ↓ (LN)</td><td>ND</td><td>= (BWL, S)</td><td>↓ (S)</td><td>(17)</td></tr><tr><td>ZIKV</td><td>FP</td><td>ND</td><td>ND</td><td>= (CS, S)</td><td>↓ (S)</td><td>(67)</td></tr><tr><td>IP</td><td>ND</td><td>ND</td><td>ND</td><td>= (brain, eye) ↑ (brain stem)</td><td>↓ (S)</td><td>(62)</td></tr><tr><td>IV</td><td>= (serum)</td><td>ND</td><td>ND</td><td>= (S)</td><td>↓ (S)</td><td>(14)</td></tr><tr><td>IN</td><td>Time dependent (brain)</td><td>↑ (brain)</td><td>↑ (brain)</td><td>= (brain, cornea, serum, trigeminal ganglia)</td><td>↑ (BWL)</td><td>(69)</td></tr><tr><td>C</td><td>ND</td><td>↑ (brain)</td><td>↑ (brain stem)</td><td>= (brain, cornea, serum, trigeminal ganglia)</td><td>↓ (S)</td><td>(64)</td></tr><tr><td rowspan="3">Herpesviridae</td><td>HSV-1</td><td></td><td></td><td></td><td>= (brain, cornea, serum, trigeminal ganglia)</td><td>↓ (S)</td><td>(63)</td></tr><tr><td>MGHV</td><td>IN</td><td>ND</td><td>ND</td><td>↑ (lung)</td><td>ND</td><td>(75)</td></tr><tr><td>IAV</td><td>IN</td><td>ND</td><td>ND</td><td>↑ (lung)</td><td>= (S, BWL)</td><td>(72)</td></tr></tbody></table>
<table><thead><tr><th></th><th></th><th></th><th>IN</th><th>↓ (BAL)</th><th>ND</th><th>↑ (lung)</th><th>↓ (S, BWL)</th><th>(76)</th></tr></thead><tbody><tr><td rowspan="3">Paramyxoviridae</td><td rowspan="3">hMPV<br>SeV</td><td>IN</td><td>↓ (lung)</td><td>ND</td><td>↑ (lung)</td><td>ND</td><td>(77)</td></tr><tr><td>IN</td><td>↓ (lung)</td><td>ND</td><td>↑ (lung)</td><td>↓ (S)</td><td>(78)</td></tr><tr><td>IN</td><td>ND</td><td>↑ (lung)</td><td>↑ (lung)</td><td>ND</td><td>(25)</td></tr><tr><td rowspan="3">Picornaviridae</td><td rowspan="3">EMCV</td><td>IP</td><td>↓ (serum)</td><td>ND</td><td>ND</td><td>↓ (S)</td><td>(16)</td></tr><tr><td>IP</td><td>= (serum)</td><td>ND</td><td>ND</td><td>↓ (S)</td><td>(14)</td></tr><tr><td>IV</td><td>↓ (serum)</td><td>ND</td><td>ND</td><td>↓ (S)</td><td>(49)*</td></tr><tr><td rowspan="2">TMEV</td><td>IC</td><td>ND</td><td>ND</td><td>↑</td><td>↓ (S)<br>↑ (BWL)</td><td>(58)</td></tr><tr><td>IN</td><td>ND</td><td>ND</td><td>= (brain)</td><td>↑ (CS)</td><td>(72)</td></tr><tr><td>Peribunyaviridae</td><td>OROV</td><td>FP</td><td>ND</td><td>ND</td><td>ND</td><td>=<br>(BWL, S)</td><td>(66)</td></tr><tr><td rowspan="3">Togaviridae</td><td rowspan="2">CHIKV</td><td>FP</td><td>↓ (foot)<br>= (blood)</td><td>= (blood)</td><td>= (serum, foot)</td><td>= (S)</td><td>(68)</td></tr><tr><td>ID</td><td>ND</td><td>ND</td><td>=</td><td>= (S)</td><td>(18)</td></tr><tr><td>SINV</td><td>IC</td><td>↓ (brain)</td><td>↑ (brain, spinal cord)</td><td>= (brain)</td><td>= (S)<br>↑ (CS, BWL)</td><td>(70)</td></tr></tbody></table>

685 C: corneal infection; BAL: Bronchoalveolar lavage; BWL: body weight loss; CHIKV: Chikungunya virus;
686 CS: clinical signs; DENV dengue virus; EMCV: encephalomyocarditis virus; FP: footpad; hMPV: human
687 metapneumovirus; HSV-1: herpes simplex virus 1; IAV: influenza A virus; IC: intracranial; ID:
688 intradermal; IN: intranasal; IP: intraperitoneal; IV: intravenous; LN: lymph nodes; MGHV: mouse
689 gammaherpes virus; ND: not determined; O: oral; OROV: Oropouche virus; S: survival; SeV: Sendai
690 virus; SINV: Sindbis virus; SP: spinal cord; TMEV: Theiler's murine encephalomyelitis virus; WNV: West
691 Nile virus; ZIKV: Zika virus; ↑: higher in *Irf3* KO than in WT mice; ↓: lower in *Irf3* KO than in WT mice;
692 =: not significantly different between *Irf3* KO and WT mice. All studies used *Irf3*<sup>tm1Ttg</sup> mice (16), except
693 * which used *Irf3*<sup>tm2.1Ttg</sup> (49).
695

696

**Figure Legends**

697

**Figure 1: Structure and functions of IRF3.** (A) Organization of the mouse *Irf3* gene. Thick boxes represent the 7 exons. Thin boxes represent untranslated exonic regions. Numbers show the corresponding amino acids of the protein at the start of each exon and at the end of the coding sequence. (B) Protein organization showing the structural domains. DBD: DNA binding domain.

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NLS: nuclear localization signal. NES: nuclear export signal. RIKA: domain associated with repression of IRF3-mediated NF-κB activity. RIPA: domain associated with RLR-induced IRF3-mediated pathway of apoptosis. IAD: IRF association domain. SRR: serin-rich region. Black outlines indicate the corresponding exons. (C) Representations of the three functions of IRF3: transcriptional activation (green arrow, center), repression of IRF3-mediated NF-κB activity (RIKA; blue arrow, left) activated by TLR and RLR, and RLR-induced IRF3-mediated pathway of apoptosis (RIPA; red arrow, right). P: phosphorylation. U: ubiquitination. C: cytochrome C.

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**Figure 2: Biphasic induction of IFN-I by IRF3 and IRF7.** During the "early phase", recognition of pathogen-associated molecular patterns (PAMP) by PRR (TLR and RLR) leads to the activation by phosphorylation (P) of IRF3 which is present in the cytosol under basal conditions, and to the expression of *Ifnb1*. Once secreted, IFN-I binds the IFNAR receptor which activates the STAT1/2 proteins and leads to the expression of ISGs including *Irf7*. In the "late phase", IRF7 is present in the cytosol. PRRs activation induce the phosphorylation of IRF7 leading to expression of the IFNα genes, which in turn enhance the expression of *Irf7* in a positive-feedback loop.

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<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>