Articles

https://doi.org/10.1038/s41556-019-0416-0

Mitochondria-localised ZNFX1 functions as a

dsRNA sensor to initiate antiviral responses
through MAVS
Yao Wang1, Shaochun Yuan1, Xin Jia1, Yong Ge1, Tao Ling1, Meng Nie1, Xihong Lan1, Shangwu Chen1 and
Anlong Xu 1,2*

In the past two decades, emerging studies have suggested that DExD/H box helicases belonging to helicase superfamily 2
(SF2) play essential roles in antiviral innate immunity. However, the antiviral functions of helicase SF1, which shares a con-
served helicase core with SF2, are little understood. Here we demonstrate that zinc finger NFX1-type containing 1 (ZNFX1), a
helicase SF1, is an interferon (IFN)-stimulated, mitochondrial-localised dsRNA sensor that specifically restricts the replication
of RNA viruses. Upon virus infection, ZNFX1 immediately recognizes viral RNA through its Armadillo-type fold and P-loop
domain and then interacts with mitochondrial antiviral signalling protein to initiate the type I IFN response without depending
on retinoic acid-inducible gene I-like receptors (RLRs). In short, as is the case with interferon-stimulated genes (ISGs) alone,
ZNFX1 can induce IFN and ISG expression at an early stage of RNA virus infection to form a positively regulated loop of the well-
known RLR signalling. This provides another layer of understanding of the complexity of antiviral immunity.

T
he innate immune system is the first line of host defence against
pathogen invasion. Upon viral infection, virus-derived nucleic
acids are mainly sensed by two cytosolic nucleic acid sen-
sors, the DNA sensor cyclic guanosine monophosphate–adenosine
monophosphate synthase (cGAS)1,2 and the retinoic acid-inducible
gene I (RIG-I)-like receptors (RLRs), including RIG-I and MDA53–6.
In addition to these well-known nucleic acid sensors, many other
nucleic acid sensors, including several DExD/H box helicases, have
been identified in a wide range of cell types to supplement the
functions of RLRs and cGAS7. For example, DDX60 is involved in
RIG-I-dependent and -independent antiviral responses8, DDX1,
DDX21 and DHX36 helicases form a complex with TRIF to sense
dsRNA in dendritic cells9, DHX9 mediates NLRP9b recognition of
dsRNA stretches and forms inflammasome complexes to promote
the maturation of interleukin (IL)-18 and gasdermin D-induced
pyroptosis10 and NLRP6 binds viral RNA via DHX15 and interacts
with mitochondrial antiviral signalling protein (MAVS) to induce
type I/III interferons (IFNs)11. These emerging studies highlight
the crucial roles of DExD/H box helicases, which belong to helicase
superfamily 2 (SF2), in antiviral immune responses. However, as a
different superfamily of many other helicases, helicase SF1 is rarely
characterized in sensing pathogenic nucleic acids, although these
proteins share a conserved core composed of two tandem RecA
domains with ATPase activity and RNA binding ability12–14.
After binding viral-derived nucleic acids to cytosolic viral sen-
sors, two adaptors, MAVS and STING, can be recruited to induce
the production of type I IFNs and a number of interferon stimulated
genes (ISGs) with antiviral activities15–24. For example, the myxovi-
rus resistance 1 (Mx1) gene product is the inhibitor of virus entry25,
the interferon-induced transmembrane (IFITM) family prevents
infection before virus can traverse the lipid bilayer of the cell26, PKR
impedes viral translation through phosphorylation of eIF2a elon-
gation factors27, while tetherin causes the retention of virions on
infected cell surfaces28,29. In addition to inhibiting a specific viral life
cycle stage, some ISGs can enhance innate pathogen-sensing capa-
bilities or positively regulate IFN signalling24. For example, poly-
glutamine binding protein 1 binds to reverse-transcribed HIV-1
DNA and interacts with cGAS to activate the type I IFN response30,
RNF128 functions as an E3 ligase for K63-linked ubiquitination
and activation of TBK131 and human IFN-inducible oligoadenyl-
ate synthetase-like protein mediates RIG-I activation by mimicking
polyubiquitin32. Thus, ISGs exert diverse functions at multiple levels
of antiviral immunity.
To determine the antiviral functions of rarely studied helicase
SF1 and identify more ISGs, we analysed previously published
in vitro transcription-sequencing alternative polyadenylation sites
(IVT-SAPAS) data of vesicular stomatitis virus (VSV)-infected
monocyte-derived macrophages (MDMs) and identified ZNFX1
(a member of helicase SF1) as an ISG and mitochondrial-local-
ised dsRNA sensor33. ZNFX1 can initiate IFN and ISG expression
immediately upon RNA virus infection by sensing viral RNAs and
interacting with MAVS, without depending on RLRs. The identi-
fication of ZNFX1 not only sheds light on the functions of RNA
helicase SF1 in antiviral immunity, but also provides a target for
antiviral therapy.
Results
Viral infection induces ZNFX1 expression. To identify undis-
covered molecules involved in antiviral innate immunity, we ana-
lysed our previously published IVT-SAPAS data of VSV-infected
MDMs and found numerous genes with changes in their expres-
sions (Extended Data Fig. 1a)33. By comparing VSV-infected human
MDM data and mouse peritoneal macrophage data, we found
some common genes, which were upregulated more than three-
fold, including FFAR2, ZEB2, TIPARP, PTGS2, CMPK2, ZNFX1
and CSRNP1. We then knocked down these genes in A549 cells and

State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, School of Life Science, Sun Yat-sen University,
1

Guangzhou, China. 2School of Life Science, Beijing University of Chinese Medicine, Beijing, China. *e-mail: lssxal@mail.sysu.edu.cn

1346 Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology

NATURE CELL BIoLoGy
subsequently infected the cells with VSV-eGFP (enhanced green
fluorescence protein). Flow cytometry to detect the percentage
of GFP-positive cells showed that knocking down ZNFX1, whose
antiviral roles have never been reported, facilitated VSV infection
(Extended Data Fig. 1b). Many previous studies have suggested that
DExD/H box RNA helicases, belonging to RNA helicase SF2, play
diverse roles in antiviral immune responses7. ZNFX1, a representa-
tive of RNA helicase SF1, contains an Armadillo-type fold (ARM)
domain, a P-loop helicase domain and a zinc finger (ZF) domain.
Because genes from helicases SF1 and SF2 share a conserved
core structure, characterized by two tandem RecA domains, we
proposed that SF1 members may also play important roles in antivi-
ral immunity, as previously suggested13.
To test this hypothesis, we analysed the gene expression pro-
files of SF1 and SF2 using IVT-SAPAS data and found a number
of genes in these two subfamilies with changes in expression upon
VSV infection (Fig. 1a). Flow cytometry results showed that knock-
ing down MOV10, HELZ2, UPF1 and ZNFX1 in A549 cells led to
markedly enhanced VSV replication, whereas SEXT knockdown
caused the opposite result (Extended Data Fig. 1c,d). MOV10, UPF1
and SEXT have been implicated previously in antiviral immunity,
validating our screening approach based on RNAi and viral infec-
tion, and suggesting that ZNFX1 is probably an undiscovered
antiviral molecule34–36.
To obtain more insight into the role of ZNFX1 in antiviral
responses, qRT–PCR or immunoblot analyses were conducted to
obtain the upregulated expression pattern of ZNFX1 in A549 cells,
peripheral blood mononuclear cells (PBMCs) or mouse embryonic
fibroblast (MEF) cells upon VSV infection or poly(I:C) stimula-
tion (Fig. 1b–d). The abundance of Znfx1 in mouse spleen and lung
and the upregulation in immune-related tissues including thymus,
lymph node, spleen and lung after intravenous injection of VSV
were also demonstrated by qRT–PCR (Fig. 1e). We also conducted
statistical analyses of previously collected microarray data sets (GD
S4231/4214/1271/3210/6082/6063) and found that RNA levels
of Znfx1 were significantly higher in patients with HIV infection
and monkeys with encephalitis caused by simian immunodefi-
ciency virus (SIV) when compared with healthy controls (Fig. 1f,g).
Moreover, the transcription of Znfx1 is significantly increased after
different virus infections in different cell types, including macro-
phages, dendritic cells and epithelial cells (Extended Data Fig. 1e,f).
These data all indicate that ZNFX1 may be an antiviral molecule.
ZNFX1 is an IFN-stimulated gene. Because viral infection led to
the enhancement of ZNFX1 expression at both mRNA and protein
levels, we next examined whether ZNFX1 is an ISG by incubating
A549 cells with human recombinant IFN-α and IFN-β. The results
of qRT–PCR and immunoblot analyses showed that the expression
of ZNFX1 could be induced by IFN-α and IFN-β (Fig. 1h,i). In con-
trast, the induction of Znfx1 mRNA by VSV could be inhibited by
the JAK1 inhibitor ruxolitinib and was almost abolished in Ifnar1−/−
A549 cells (Fig. 1j,k).
To identify which nucleotides and region are important in deter-
mining Znfx1 transcription, bioinformatics analyses using the
promoter 2.0 prediction server (http://www.cbs.dtu.dk/services/
promoter/), JASPAR (http://jaspar.genereg.net/) and CpG plot
(http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/) were per-
formed to identify the region between −1,064 and +63 of Znfx1
containing TATA box, GC box and CAAT box, which are the char-
acteristics of a promoter. Further analyses of transcription factor
binding sites revealed that the promoter of Znfx1 contains STAT1,
STAT2, IRF1 and IRF9 binding motifs in a relatively concentrated
and overlapped region, as shown in Extended Data Fig. 1g. Based on
these analyses, we constructed a Znfx1 promoter reporter construct
containing the region between −1,064 and +63 and a mutated
reporter construct with deletions of the STAT1, STAT2, IRF1 and
Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology
Articles
IRF9 binding motifs. Next, these two reporter constructs were
transfected with increasing amounts of STAT1, STAT2, IRF1 or
IRF9 expression plasmids into 293T cells. Reporter assays showed
that the expression of reporter gene could be induced by the Znfx1
promoter but not by a gene lacking the transcriptional factor bind-
ing sites in the presence of STAT1, STAT2, IRF1 or IRF9 (Fig. 1l).
In all, ZNFX1 is an ISG whose upregulation upon viral infection is
dependent on the JAK1-STAT1 cascade.
ZNFX1 is essential for innate anti-RNA virus in vitro and in vivo.
To further explore the role of ZNFX1 in antiviral immune responses,
we silenced the endogenous expression of ZNFX1 in A549 and L929
cells. The cells were then infected with VSV-eGFP and subjected
to flow cytometry analysis (Extended Data Fig. 2a,b). The results
show that knockdown of Znfx1 significantly enhanced VSV rep-
lication both in A549 and L929 cells (Fig. 2a). Meanwhile, higher
VSV titres and increased VSV-G protein were observed in Znfx1
silencing cells (Fig. 2b,c). Consistently, knockdown of Znfx1 expres-
sion significantly decreased the production of IFN-α and IFN-β
protein in A549 cells after VSV infection or poly(I:C) stimulation
(Extended Data Fig. 2c). In contrast, lower VSV mRNA and VSV
titres in A549 cells were observed when ZNFX1 was overexpressed
(Extended Data Fig. 2d).
To obtain more functional evidence for ZNFX1 in antivi-
ral responses, we employed a CRISPR-mediated genome editing
approach to knock out Znfx1 in 293T and A549 cells. Similar to the
transient silencing of Znfx1 in A549 or L929 cells, we found that
genome editing of Znfx1 in 293T and A549 cells enhanced VSV
replication (Fig. 2d and Extended Data Fig. 2e,f). Meanwhile, over-
expression of ZNFX1 in wild-type or Znfx1−/− A549 cells restricted
VSV replication (Fig. 2e). Further RNA sequencing analysis com-
paring the genes differentially expressed between wild-type and
ZNFX1-overexpressed A549 cells showed the upregulated enrich-
ment of genes involved in negative regulation of viral replication
and signal transduction of type I IFN (Fig. 2f). Interestingly, virus
mRNA levels were increased in Znfx1 knockdown A549 cells with
EMCV and influenza A virus (H1N1) infection, but not signifi-
cantly changed upon herpes simplex virus type 1 (HSV-1) infection,
suggesting a broad anti-RNA-viral spectrum of ZNFX1 (Extended
Data Fig. 2g).
To investigate the physiological role of ZNFX1 in vivo, we gener-
ated Znfx1−/− C57BL/6J mice using CRISPR/Cas system (Extended
Data Fig. 3a–c). Znfx1−/− mice were viable, without obvious physi-
ological or behavioural abnormalities. Six- to eight-week-old mice
were used in this study. First, BMDMs from wild-type and Znfx1−/−
mice were isolated and infected with VSV, EMCV, H1N1 or HSV-1.
Compared to wild-type counterparts, Znfx1−/− BMDMs exhibited
lower IFN-α or IFN-β secretion after RNA virus infection, includ-
ing VSV, EMCV and H1N1 but not HSV-1 (Fig. 2g and Extended
Data Fig. 2h). Consistently, higher virus mRNA levels were detected
in VSV, EMCV and H1N1 infected Znfx1−/− BMDMs but not in
HSV-1 infected cells (Fig. 2h). Compared to wild-type counterparts,
Znfx1−/− MEF and A549 cells exhibited lower IFN-β secretion after
RNA virus infection of VSV and EMCV (Fig. 2i,j).
To evaluate the importance of ZNFX1 in host defence against
viral infection in vivo, wild-type and Znfx1−/− mice were injected
with VSV through the caudal vein. Enhancement of VSV replication
in lung and liver of Znfx1−/− mice was observed when compared with
wild-type mice (Fig. 3a). With reduced expression of Ifnb1 in lung
and liver, lower secretion of IFN-α and IFN-β in serum was detected
in Znfx1−/− mice (Fig. 3b,c). Notably, no difference in IFN-α and
IFN-β in serum was observed between Znfx1−/− and wild-type mice
after HSV-1 infection (Fig. 3c). Interestingly, one day post infec-
tion with VSV, 80% of Znfx1−/− mice suffered from conjunctivitis,
which is the early symptom of VSV infection (Fig. 3d). In addition,
more infiltration of inflammatory cells into the lungs of Znfx1−/−
1347
Articles NATURE CELL BIoLoGy

a MDM b c
VSV: 0 2 4 8 16 24 h 50 VSV: 0 4 8 16 24 h Poly(I:C) 0 4 8 16 24 h

expression of Znfx1
Relative mRNA
40 Znfx1 Znfx1

A549

A549
30
20 β-actin β-actin

10
0 Znfx1 Znfx1

MEF

MEF
VSV : – + + + + – – – – –
Poly(I:C) – – – – – – + + + + β-actin β-actin
Time (h): 0 4 8 16 24 0 4 8 16 24

d VSV: 0 4 8 16 24 h e
245 kDa Spleen Thymus
IB: ZNFX1 Lung Lymph node
A549

Kideny Spleen
Heart Lung
IB: GAPDH 35 kDa Bowel Bowel
Liver Muscle
Thymus Kidney
Lymph node Brain Virus infection
IB: ZNFX1 245 kDa
PBMC

Brain Heart Uninfected

–2 0 2 Muscle Liver
SF2: DEAD-box IB: GAPDH 35 kDa 0 30 60 90 120 0 5 10 15 20 25
MDM Znfx1 mRNA (relative) Znfx1 mRNA (relative)
VSV: 0 2 4 8 16 24 h
f 2.0 P = 0.0123 1.0 P = 0.0004 Uninfected (UI) g 11 P = 3.77 × 10–15 10 P = 4.50 × 10–12 Uninfected (UI)
transformed count

transformed count

transformed count

transformed count
HIV infection SIV infection
1.5 10 8
0.5
1.0
Znfx1

Znfx1
Rig-I

Rig-I
9 6
0.5
–1.5 0 1.5 0
0 8 4
SF1 RNA helicase
–0.5 –0.5 7 2
UI HIV UI HIV UI SIV UI SIV
h 15
expression of Znfx1

i
Relative mRNA

IFN-α: 0 4 8 16 24 h IFN-β: 0 4 8 16 24 h
10
IB: ZNFX1 245 kDa IB: ZNFX1 245 kDa

5
A549

IB: RIG-I 100 kDa IB: RIG-I 100 kDa

0
IFN-α: – + + + + – – – – – IB: β-actin 45 kDa IB: β-actin 45 kDa
IFN-β: – – – – – – + + + +
Time (h): 0 4 8 16 24 0 4 8 16 24

j UT k WT l
wt Znfx1-Luc
Rux Ifnar –/–
ΔZnfx1 promotor-Luc
P = 0.0065 –6 P = 0.0003 P = 7.44 × 10–6
15 P = 1.69 × 10
15 4 P = 0.0009 4 10 80 P = 4.40 × 10–6
expression of Znfx1

expression of Znfx1

P = 0.0043 P = 0.0003
Relative mRNA

Relative mRNA

Rel. Znfx1-Luc

Rel. Znfx1-Luc

Rel. Znfx1-Luc

Rel. Znfx1-Luc

3 3 8 60
10 10
activity

activity

activity

activity

6
2 2 40
4
5 5
1 1 2 20
0 0 0 0 0 0
VSV: 0 4 8h VSV: 0 4 8h EV + – – – + – – – EV + – – – + – – – EV + – – – + – – – EV + – – – + – – –
STAT1 – – STAT2 – – IRF1 – – IRF9 – –

Fig. 1 | Viral infection induces Znfx1 expression, an IFN-stimulated gene. a, MDMs were infected with VSV, then IVT-SAPAS was performed. The heatmap
shows an abundance change of RNA helicases SF1 and SF2 in virus-infected MDMs. b, A549 cells were challenged with VSV or stimulated with poly(I:C)
for the indicated time. The mRNA levels were analysed by qRT–PCR. c, A549 and MEF cells were challenged with VSV or stimulated with poly(I:C) for the
indicated time. The mRNA levels were analysed by RT–PCR. d, Immunoblot (IB) analysis of ZNFX1 protein in A549 cells and peripheral blood mononuclear
cells (PBMCs) upon VSV infection at the indicated time points. e, Left: Distribution of Znfx1 in various mice tissues was determined using qRT–PCR analysis;
the transcription of Znfx1 in muscle was set to 1, values of other tissues are expressed as the ratio compared with muscle. Right: Znfx1 mRNA expression
change in various mice tissues following intravenous injection of VSV determined by qRT–PCR. f, RNA levels of Znfx1 are significantly higher in patients
with HIV infection compared with uninfected controls (UI) (n = 9 independent samples for uninfected controls, n = 14 independent samples for HIV
infection). g, RNA levels of Znfx1 and Rig-I in encephalitis monkeys infected with simian immunodeficiency virus (SIV) compared with healthy controls
(n = 18 independent samples). h, Znfx1 expression was measured by qRT–PCR in A549 cells after IFN-α or IFN-β treatment for the indicated time points.
i, Immunoblot analysis of ZNFX1 and RIG-I expression in A549 cells treated with human recombinant IFN-α or IFN-β for the indicated time points. j, Znfx1
expression was measured by qRT–PCR in A549 cells after ruxolitinib (Rux) pretreatment for 2 h, then infected with VSV for time points. k, Znfx1 expression
was measured by qRT–PCR in wild-type (wt) and Ifnar1−/− cells after VSV infection. l, Luciferase activities determined from 293T cells transfected with Znfx1
promoter reporter (wt Znfx1-Luc) or its mutation (ΔZnfx1 promotor-Luc) together with increasing amounts of Flag-STAT1, STAT2, IRF1, IRF9 expressing
plasmids. For c,d,i, experiments were repeated at least three times independently, with similar results obtained. For b,e,h,j–l, n = 3 independent experiments.
Means ± s.d. are shown, and P values were calculated using two-tailed unpaired Student’s t-test.

1348 Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology

NATURE CELL BIoLoGy Articles
a VSV-eGFP (MOI: 2) 6 h VSV-eGFP (MOI: 5) 6 h c VSV-eGFP
d
VSV-eGFP (MOI: 0.5) 12 h VSV-eGFP (MOI: 1) 12 h A549
65 kDa

VSV-GFP+ (% of total)
–5
P = 0.0013 P = 1.23 × 10–6 IB: VSV-G P = 7.34 × 10
80 100 100 WT

A549
A549 VSV-GFP+

P = 0.0028
80 80 Rig i –/–
(% of total)

(% of total)
60

VSV-GFP+
P = 4.60 × 10–6 IB: GAPDH 35 kDa
60 60 Mda5 –/–

L929
40 65 kDa Znfx1–/–
40 IB: VSV-G 40

L929
20 20 20
0 0 IB: GAPDH 35 kDa 0
Uninfect. VSV
N X1

N X1

1
ZN VS

ZN VS

ZN VS

ZN VS
C STM

C STM

C STM
A l

A l

C STM
A l

A l
M tro

M tro

M tro

M tro

1
S
FX

FX

TM

M l
tro

FX
AV
F

F
on

on

on

on

on
-S
-

ZN
N

N
C
VSV-eGFP VSV-eGFP VSV-eGFP VSV-eGFP

b P = 0.0002 P = 3.19 × 10
–6 e P = 0.0003
f
30 40 80
p.f.u. ml−1 (×106)

p.f.u. ml−1 (×106)

P = 0.0023 WT Negative regulation of viral replication 4.60 × 10−8

Znfx1–/–

(% of total)
VSV-GFP+
30 60
20 Type I IFN signalling 1.60 × 10−7
A549

L929

20 40 EGFR signalling 2.50 × 10−7

10 Defence response to virus 2.60 × 10−7
10 20
TRIF-dependent TLR signalling 3.80 × 10−7
0 0 0
EV + + – + + – – 0 2 4 6 8
1
ZN VS

M rol

1
ZN VS
M trol

FX

FX

VSV – + + – + + +
t
A
A

on
on

P value (–log10)
C
C

ZNFX1 – – + – – – +
VSV-eGFP VSV-eGFP RIG-I – – – – – + –

g –6
h P = 0.0042 P = 0.0019 P = 4.24 × 10–5 P = 0.2616
P = 1.36 × 10–6

Rel. mRNA of HSV-1

Rel. mRNA of EMCV
P = 1.09 × 10

Rel. mRNA of H1N1

3,000 2,000 10,000
Rel. mRNA of VSV

2,500 2,500 8,000 WT

WT
IFN-β (pg ml−1)

IFN-β (pg ml−1)

2,000 2,000 1,500 8,000 Znfx1–/–

Znfx1–/– 6,000
2,000
1,500 1,500 6,000
4,000 1,000
1,000 1,000 1,000 4,000
500 500 2,000 500 2,000
0 0 0 0 0 0
VSV EMCV VSV EMCV H1N1 HSV-1
i
500
P = 7.89 × 10–6
800
P = 0.6519
3,000 P = 0.0001 P = 0.0006 j 1,500 P = 0.0003 P = 3.42 × 10 –6

WT

IFN-β (pg ml−1)

IFN-β (pg ml−1)
IFN-β (pg ml−1)

IFN-β (pg ml−1)

400 600 WT
2,000 Rig i –/– 1,000
300 Mda5 –/– Rig i –/–
400
200 Znfx1–/– Mda5 –/–
1,000 500
100 200 Znfx1–/–
0 0 0 0
H1N1 HSV-1 Uninfect. VSV EMCV Uninfect. VSV EMCV

Fig. 2 | ZNFX1 deficiency impairs cellular antiviral RNA response. a, FACS analysis of A549 and L929 cells transfected with control small interfering
(si)RNA or the indicated siRNAs followed by VSV-eGFP infection at different multiplicity of infection (MOI) (n = 4 independent experiments). b, Plaque
assay of VSV titres in cell supernatants after VSV infection for 12 h (n = 4 independent experiments). c, Immunoblot analysis of VSV-G protein expression
in A549 and L929 cells infected with VSV for 12 h. The experiments were repeated three times, independently, with similar results. d, FACS analysis
of wild-type, Znfx1−/−, Rig-I−/− or Mda5−/− cells infected by VSV-eGFP. e, FACS analysis of wild-type and Znfx1−/− A549 cells transfected with RIG-I,
ZNFX1 expressing plasmids or empty vector (EV) following VSV-eGFP infection. f, Gene ontology analysis of the pathways among genes significantly
upregulated between ZNFX1 overexpressed cells and wild-type A549 cells. The Kolmogorov–Smirnov test was used to calculated P values. g, Enzyme-
linked immunosorbent assay (ELISA) of IFN-β in supernatants of bone marrow-derived macrophages (BMDMs) from wild-type and Znfx1−/− mice infected
with VSV, encephalomyocarditis virus (EMCV), influenza A virus (H1N1) or herpes simplex virus type 1 (HSV-1) for 16 h (n = 5 independent experiments).
h, Viral mRNA transcripts were analysed in VSV, EMCV, H1N1 and HSV-1 infected wild-type and Znfx1−/− BMDMs by qRT–PCR analysis. i, ELISA of IFN-β in
supernatants of MEFs from wild-type and Znfx1−/−, Rig-I−/−, Mda5−/− mice infected with VSV and EMCV. j, ELISA of IFN-β in supernatants of wild-type and
Znfx1−/−, Rig-I−/−, Mda5−/− A549 cells infected with VSV and EMCV. For d,e,h–j, n = 3 independent experiments. All data are presented as mean ± s.d.
P values were calculated using two-tailed unpaired Student’s t-test.

mice following VSV infection was observed (Fig. 3e) and Znfx1−/− the activation of Ifnb1 promoter triggered by VSV, two types of
mice were less resistant to VSV infection in overall survival assays poly(I:C) or poly(dA:dT) in a dose-dependent manner (Fig. 4b,c).
(Fig. 3f). These data suggest that Znfx1 deficiency has impaired In contrast, knockout of Znfx1 decreased VSV-induced activation of
innate immune responses against RNA virus infection by producing Ifnb1 reporter and ISRE reporter in 293T cells (Fig. 4d). Meanwhile,
less type I IFNs in vivo. In addition, we suggested that ZNFX1 exerts transcription of Ifnb1 and ISGs was induced in a dose- and time-
its antiviral properties specifically on RNA viruses. dependent manner when ZNFX1 was overexpressed alone in A549
cells but reduced in the absence of ZNFX1 (Fig. 4e and Extended
ZNFX1 initiates IFN-based antiviral responses. To investigate the Data Fig. 3d,e), while protein expression of a number of ISGs was
role of ZNFX1 in the IFN signalling, luciferase reporter assays were reduced in Znfx1−/− A549 and MEF cells when compared with wild-
conducted by transiently transfecting the A549 cells with increas- type cells, respectively (Fig. 4f). Consistently, the phosphorylation
ing amounts of ZNFX1 expression plasmid and Ifnb1-reporter or of TBK1 and IRF3 in Znfx1−/− macrophages upon VSV infection
IFN-stimulated response element (ISRE) reporter plasmids fol- was lower than that in wild-type macrophages (Fig. 4g). The dimer-
lowed with or without VSV challenge. The results showed that ization of IRF3 was also lower in VSV-infected Znfx1−/− 293T cells
overexpression of ZNFX1 activated the Ifnb1 promoter and ISRE than that in wild-type cells (Fig. 4h). Notably, restricted replication
reporter slightly (Fig. 4a). Moreover, ZNFX1 significantly enhanced of VSV was observed in wild-type but not in Ifnar1−/− A549 cells

Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology 1349

Articles NATURE CELL BIoLoGy

a b P = 0.0005
15 15 200 800 WT

replicates (fold ×103)

replicates (fold ×103)

P = 0.0008 WT –/–
P = 0.0009 Znfx1
P = 8.69 × 10–6 Znfx1
–/–

Rel. mRNA of

Rel. mRNA of
150 600
VSV RNA

VSV RNA
10 10

Ifnb1

Ifnb1
100 400
5 5
50 200

0 0 0 0
PBS VSV PBS VSV PBS VSV PBS VSV
Lung Liver Lung Liver

c WT Znfx1–/–
d
P = 3.12 ×10–9 VSV injection 24 h
P = 0.1361
1,800
IFN-α (pg ml−1) 800 –11
P = 7.65 ×10 250 200 P = 0.9430

IFN-α (pg ml−1)

IFN-β (pg ml−1)
IFN-β (pg ml−1)

600 200 150

1,200
150
400 100
100
600
200 50 50

0 0 0 0
PBS VSV PBS VSV PBS HSV-1 PBS HSV-1 WT Znfx1–/–

e Znfx1+/+ Znfx1–/–
f
Znfx1+/+
100 Znfx1–/–

80
Survival (%)
PBS

60
40
P = 0.0283
20
0
0 1 2 3 4 5
Time after injection (days)
VSV

Fig. 3 | ZNFX1 is essential for host defence against RNA virus in mice. a, VSV mRNA transcripts were analysed by qRT–PCR in the lung and liver from
wild-type and Znfx1−/− mice with VSV infection by intravenous injection (2 × 107 p.f.u. per g body weight, n = 4 mice per group). b, qRT–PCR analysis of
Ifnb1 mRNA in the lung and liver from wild-type and Znfx1−/− mice (n = 4 mice per group). c, IFN-α and IFN-β production in sera from wild-type mice and
Znfx1−/− mice after intravenous injection with VSV and HSV-1 (8 or 4 mice per group). d, One day post infection with VSV, Znfx1−/− mice suffered from
conjunctivitis, unlike wild-type mice. e, Haematoxylin & eosin staining of lung sections from wild-type and Znfx1−/− mice infected with VSV for 24 h. Scale
bars, 80 μm. The experiments were repeated three times, independently, with similar results. f, Survival of wild-type and Znfx1−/− mice (n = 10 mice per
group) after intravenous injection of VSV (2 × 107 p.f.u. per g body weight). Data in a–c are presented as mean ± s.d. Statistical differences were detected
with two-tailed unpaired Student’s t-tests in a–c. A Gehan–Breslow–Wilcoxon test was applied in f.

(Fig. 4i). All these data suggest that ZNFX1 is involved in antiviral Pulldown assays were performed to show that ZNFX1 and RIG-I
immune responses by inducing type I IFN production and its anti- proteins bind to poly(I:C) but not poly(C) (Fig. 5g). Additionally,
viral property also depends on IFN signalling. cGAS but not ZNFX1 binds to both single- and double-stranded
DNA (ISD, a 45 bp double-stranded DNA; Fig. 5h). Competition
ZNFX1 directly binds to viral RNA. Many genes belonging to experiments revealed the preferable binding of ZNFX1 proteins to
RNA helicase SF2 have been identified as RNA virus sensors that HMW poly(I:C), but not LMW poly(I:C), poly(dA:dT) or 5′ppp-
bind viral RNA, so we assumed that ZNFX1 belonging to SF1 may dsRNA (Fig. 5i). To map the poly(I:C)-binding sites of ZNFX1, we
also bind viral RNA as an RNA sensor. To verify this hypothesis, we prepared truncated versions of ZNFX1 and performed poly(I:C)
first overexpressed ZNFX1 in 293T cells and then performed pull- pulldown experiments. The results showed that the ARM and
down and qRT–PCR assays to determine the high binding ability of P-loop domains, but not the ZF domain, were required for the
ZNFX1 with VSV-RNA (Fig. 5a,b). A similar assay also detected the binding of ZNFX1 to poly(I:C) (Fig. 5j). Meanwhile, we observed
binding ability of endogenous ZNFX1 to VSV, EMCV and Senda enhanced oligomerization of ZNFX1 after viral infection (Fig. 5k,l).
virus (SeV) RNAs in A549 cells (Fig. 5c). RNA immunoprecipita- Thus, ZNFX1 functions as an RNA virus sensor, which prefers to
tion (RIP)-sequencing analysis by isolating RNA from immunopre- bind the long RNA molecules.
cipitates with antibodies against endogenous ZNFX1 also mapped
ZNFX1-associated RNA to VSV genome (Fig. 5d), preferentially to ZNFX1 localises to mitochondria and interacts with MAVS. To
a segment of VSV strand around 6,000–8,000 nt (Fig. 5e). Notably, determine the subcellular localisation of ZNFX1, we separated
when ZNFX1-associated RNA was transfected into A549 cells, VSV-infected cells into cytoplasmic and nuclear fractions, and
induced transcription of Ifnb1 and induced expression of Ifnb1 pro- detected ZNFX1 protein mainly in the cytoplasm (Fig. 6a, upper
moter-based reporter were observed by qRT–PCR and luciferase panel). Previous studies about ARM-containing proteins, ALEX3
activity analyses (Fig. 5f). and SARM1, have indicated these proteins to be mitochondrially
To further assess the nature of viral RNA bound by ZNFX1, we localised. Thus, we next determined, by immunoblot analyses,
first incubated nucleic acid analogue linked beads with a cells lysis whether ZNFX1, an ARM-containing protein, can also localise to
of 293T overexpressed with Flag-tagged ZNFX1, RIG-I or MAVS. this immune-related organelle37–39. The results show the increased

1350 Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology

NATURE CELL BIoLoGy Articles
a b VSV (MOI:0.1) d
VSV (MOI:0.5) WT ZNFX1–/–
P = 0.0009 P = 0.0006 P = 0.0006 P = 0.0097
8 6 200 P = 8.29 × 10
–5 8 P = 0.0140 400

Rel. IFN-β-Luc
Rel. IFN-β-Luc

Rel. IFN-β-Luc
Rel. ISRE-Luc
Rel. ISRE-Luc
P = 0.0006
6 150 6 300
4

activity

activity
activity

activity
activity
4 100 4 200
2
2 50 2 100
0 0 0 0 0
EV + – – + – – EV + + – – + + – – + + – – + + – – VSV – –
ZNFX1 – – VSV – + + + – + + + – + + + – + + +
ZNFX1 – – – – – – – – P = 0.0060
245 kDa 245 kDa 150
IB: α-Flag

Rel. ISRE-Luc
100

activity
IB: GAPDH 35 kDa 35 kDa

50
c –5 P = 0.0002
P = 0.0001 P = 0.0038 P = 5.80 × 10 15 0
120
VSV – –
Rel. IFN-β-Luc

90 Rel. ISRE-Luc P = 0.0040 P = 0.0028 Poly(I:C) (HMW)

10
activity
activity

Poly(I:C) (LMW)
60
Poly(dA:dT)
5
30
e
0 0 ZNFX1: ZNFX1: 12 24 36 h
ZNFX1 – – – – – – – – – – – – Ifnb1 Ifnb1
Poly(I:C) (HMW) – + + + – – – – – – – – – + + + – – – – – – – –
Poly(I:C) (LMW) – – – – – + + + – – – – – – – – – + + + – – – – Rig-I Rig-I
Poly(dA:dT) – – – – – – – – – + + + – – – – – – – – – + + +
Ifi44 Ifi44
IB: α-Flag 245 kDa 245 kDa
Isg15 Isg15

IB: GAPDH 35 kDa 35 kDa Ifit1 Ifit1

Ifit2 Ifit2
f –/–
g Peritoneal macrophage
WT ZNFX1 Ifit3 Ifit3
VSV 0 2 4 8 0 2 4 8h WT Znfx1–/– Ifih1 Ifih1
IB: ZNFX1 245 kDa VSV 0 1 2 4 0 1 2 4h
Oas2 Oas2
IB: p-IRF3 45 kDa
IB: MDA5 135 kDa Mx1 Mx1

IB: RIG-I 100 kDa IB: IRF3 45 kDa Irf7 Irf7

A549

Znfx1 Znfx1
IB: Mx1 75 kDa IB: p-TBK1 75 kDa

65 kDa 0 5 10 4 8 12
IB: OAS1 IB: TBK1 75 kDa log2
(fold change)
IB: GAPDH 35 kDa
IB: GAPDH 35 kDa
i
WT Znfx1 –/– WT Ifnar1–/–
h P = 0.9733
VSV-GFP+ (% of total)

VSV 0 2 4 8 0 2 4 8h WT ZNFX1 –/–

80
IB: RIG-I 100 kDa VSV: 0 2 4 8 0 2 4 8h 60 P = 0.0001
MEF

Native-gel

IRF3 dimer 40
IB: GAPDH 35 kDa
20
IRF3 monomer
0
245 kDa EV + – – + – –
IB: ZNFX1
SDS–PAGE

VSV – + + – + +
ZNFX1 – – + – – +
IB: IRF3 45 kDa

IB: GAPDH 35 kDa

Fig. 4 | ZNFX1 initiates IFN-based antiviral responses. a,b, A549 cells were transfected with IFN-β-Luc or ISRE-Luc with increasing amounts of ZNFX1
plasmid for 24 h, and then uninfected (a) or infected with VSV for 16 h before luciferase assays (b). c, Luciferase activity analysis of A549 cells that were
transfected with the indicated luciferase reporters followed by stimulation with low-molecular-weight (LMW) poly(I:C), high-molecular-weight (HMW)
poly(I:C) and poly(dA:dT) for another 16 h. d, Luciferase activity of Ifnb1 or ISRE in wild-type and Znfx1−/− 293T cells that were infected with increasing
amounts of VSV for 24 h. e, qRT–PCR analysis of Ifnb1 and ISGs in A549 cells transfected with ZNFX1-expressing plasmids with increasing amounts
(125, 250 or 500 ng per well) for 24 h (left) or with 500 ng per well for the indicated time points (right). f, Immunoblot analysis of ISGs level in wild-
type and Znfx1−/− A549 cells or RIG-I expression in wild-type and Znfx1−/− MEFs infected by VSV for the indicated time points. g, Immunoblot analysis of
phosphorylated (p-) IRF3 and (p-) TBK1 in peritoneal macrophages from wild-type and Znfx1−/− mice upon VSV infection for the indicated time points.
h, Immunoblot analysis of IRF3 dimerization in wild-type and Znfx1−/− 293T cells upon VSV infection for the indicated time points with native PAGE. i, FACS
analysis of wild-type or Ifnar1−/− A549 cells transfected with ZNFX1-expressing plasmids or empty vector (EV) for 24 h followed by VSV-eGFP infection.
For f–h, the experiments were repeated three times, independently, with similar results obtained. Data in a–e,i are from three independent experiments
(mean ± s.d.). P values were calculated using two-tailed unpaired Student’s t-test.

Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology 1351

Articles NATURE CELL BIoLoGy
a VSV infection b 30 P = 0.0002 c
P = 0.0009 P = 4.18 × 10–6 –5
P = 6.06 × 10
8 6 25

VSV RNA
20 20

EMCV RNA
Bound
6

VSV RNA

SeV RNA
4

Bound

Bound

Bound
15
10 4
2 10
ZNFX1-Flag 2 5
0
RIG-I-Flag Flag or ctrl IP 0 0 0
MDA5-Flag 245 kDa IP:

G
G

1
IB: α-Flag

FX

FX

FX
Ig

Ig
Ig
135 kDa

ZN

ZN

ZN
RNA extraction 100 kDa

ag

ag

g
FX gG

la
Test for IFN stimulatory

Fl

Fl

-F
I
1-

-I-

A5
activity Test for VSV RNA

IG

D
R
ZN

M
d VSV genome (nt) f
60 25 ng 25 ng
(+) strand 40 P = 3.96 × 10
–5 50

Rel. IFN-β-Luc
50 ng P = 0.0005 50 ng

Rel. mRNA of
40
Relative numbers

30 40
P = 0.0026

activity
20 30

Ifnb1
20 P = 0.0001
0 20
10 10
−20
0 0
−40

A
er

er
ZN -RN

ZN -RN

N
(–) strand

N
at

at
R

-R

-R
W

W
−60

1-

-I-

1-

-I-
G

A5

A5
FX

FX
IG

IG
Ig

Ig
D

D
R

R
M

M
0
0

0
0

0
00

00

00

00

,0
2,

4,

6,

8,

10

e g ZNFX1 RIG-I MAVS h

) C)
IgG
I:C

)
I: ZNFX1 cGAS

C
)

ly )
3 ly( ly(
(C

(C

Po (C
(I:
Bound VSV RNA

ZNFX1 P = 0.0009 ly o Po

ly

ly
Pulldown: P Pulldown: – S D – S D
Po

Po

Po
(IP/input)

2
245 kDa
1
IB: α-Flag 100 kDa IB: α-Flag
0 65 kDa
VSV gene: N NS M G G L L L 75 kDa
VSV strand: 0–1 K 1–2 K 2–3 K 3–4 K 4–5 K 5–6 K 6–8 K 8–10 K 245 kDa 245 kDa
WCL
WCL
IB: α-Flag 100 kDa
IB: α-Flag
65 kDa
75 kDa
i Poly(I:C)(HMW) Poly(I:C)(LMW)
Competitor – –
IP: Poly(I:C) 245 kDa j ZNFX1-FL 1 ARM P-loop NH ZF 1918
IB: anti-Flag
ZNFX1-F1 580 P-loop NH ZF 1918
Poly(dA:dT) 5’-ppp dsRNA
Competitor: – – ZNFX1-F2 1261 ZF 1918

IP: Poly(I:C) 245 kDa ZNFX1-F3 1 ARM P-loop NH 1261

IB: anti-Flag ZNFX1-F4 580 P-loop NH 1261

ZNFX1-F5 1 ARM

k l ZNFX1-Flag – + + 245 kDa

ZNFX1-Flag + + + VSV – – + 135 kDa
ZNFX1-HA – + + IP: Poly(I:C) 100 kDa
IB: α-HA 75 kDa
VSV – – +
IP: α-Flag

IB: α-HA 245 kDa IB: α-Flag

ZNFX1
245 kDa
IB: α-Flag 245 kDa aggregates
Input: 135 kDa
SDD–PAGE IB: α-HA 100 kDa
IB: α-HA 245 kDa
75 kDa
WCL

IB: α-Flag 245 kDa 245 kDa SDS–PAGE

FL-HA

F1-HA

F2-HA

F3-HA

F4-HA

F5-HA

Fig. 5 | ZNFX1 binds to viral RNA. a, Schematic of the experimental set-up for ZNFX1, RIG-I and MDA5 immunoprecipitation. b, VSV-infected 293T
cells were transfected with the indicated plasmids, immunoprecipitated and analysed by qRT–PCR to detect bound VSV-RNA. c, Endogenous ZNFX1-
immunoprecipitated RNA was analysed by qRT–PCR using VSV-, EMCV- and SeV-infected A549 cells. d, RIP-sequencing mapped endogenous ZNFX1-
associated sequences to the VSV genome. The data shown are normalized with sequences from the input sample. + and − RNA is shown in red and blue,
respectively. e, qRT–PCR analysis of endogenous ZNFX1-immunoprecipitated RNA in VSV-infected A549 cells, the x axis corresponds to the positions in
the VSV genome, includeing of five coding genes. f, ZNFX1 co-immunoprecipitated RNA was first transfected into A549 cells, then luciferase activity and
mRNA transcripts of Ifnb1 were assessed. g, Flag-tagged ZNFX1, RIG-I or MAVS plasmids were transfected into 293T cells, then whole-cell lysates
(WSL) were incubated with agarose bead-conjugated poly(C) or poly(I:C). The proteins bound to agarose beads were analysed by immunoblotting.
h, Flag-tagged ZNFX1 or cGAS plasmids were transfected into 293T cells and agarose bead-conjugated DNA was incubated with whole-cell lysates,
then the bound proteins were analysed by immunoblotting (S, single-stranded DNA; D, double-stranded DNA). i, Flag-ZNFX1 recombinant protein
was incubated with agarose beads-conjugated poly(I:C) in the absence or presence of increasing amounts (0.5, 5 and 50 μg ml−1) of unlabelled HMW-
poly(I:C), LMW-poly(I:C), poly(dA:dT) or 5′ppp-dsRNA. Bound complexes were pelleted by centrifugation and analysed by immunoblot. j, Schematics
of ZNFX1 and its serial deletion mutants. The truncated versions of HA-tagged ZNFX1 were transfected into 293T cells, then whole-cell lysates were
incubated with agarose bead-conjugated poly(I:C). Bound proteins were analysed by immunoblotting. k, Co-immunoprecipitation and immunoblot
analysis of 293T cells transfected with Flag-ZNFX1 and HA-ZNFX1. l, Immunoblot analysis of ZNFX1 in 293T cells transfected with Flag-ZNFX1 plasmids
by SDD–PAGE or SDS–PAGE assay. Data in b,c,e,f are presented as mean ± s.d. (n = 3 independent experiments). P values were calculated using two-tailed
unpaired Student’s t-test. For g–l, the experiments were repeated three times, independently, with similar results obtained.

1352 Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology

NATURE CELL BIoLoGy Articles
a Cyt. Nuc. b Mock VSV 8 h VSV 16 h
c
Mito.
VSV – + – + Proteinase K – + +

ZNFX1-Flag
IB: ZNFX1 245 kDa 1% Triton – – +
IB: ZNFX1 245 kDa
IB: Lamin B1 65 kDa
IB: MAVS 75 kDa
IB: β-actin 45 kDa
IB: COX IV 15 kDa
Cyto. Mito.
Mito.

Mito
VSV – + – +
Alkali – + +
IB: ZNFX1 245 kDa M P S
IB: ZNFX1 245 kDa
IB: β-actin 45 kDa

IB: COX IV IB: MAVS 75 kDa

Merge
15 kDa
IB: COX IV 15 kDa
d
M 88

G
ST S

Flag-adaptor
IN
M F

AV
yD
I
EV
TR

ZNFX1-HA + + + + + e IP IgG ZNFX1 IgG ZNFX1 f IP IgG MAVS IgG MAVS

IP: α-Flag 245 kDa WT Znfx1–/– WT Mavs–/–
IB: α-HA
VSV – – + – – + VSV – – + – – +
100 kDa
245 kDa IB: ZNFX1 245 kDa
IP: α-Flag * 75 kDa IB: ZNFX1
IB: α-Flag
35 kDa IB: MAVS 75 kDa IB: MAVS 75 kDa

IB: α-HA 245 kDa IB: ZNFX1 245 kDa IB: ZNFX1 245 kDa
WCL

WCL
WCL

100 kDa IB: MAVS 75 kDa IB: MAVS 75 kDa

* 75 kDa
IB: α-Flag IB: GAPDH 35 kDa IB: GAPDH 35 kDa

35 kDa
h j
g Ifnb1
ZNFX1-Flag

IP IgG MAVS Rig-I

VSV 0 0 4 8 12 h
Ifi44
245 kDa 11
IB: ZNFX1

log2(fold change)
Ifit1
75 kDa
MAVS-GFP

IB: MAVS Ifit2

65 kDa 6.5
IgG (H) Ifit3
Ifih1
2
IB: ZNFX1 245 kDa Oas2
Isg15
Merge
WCL

IB: MAVS 75 kDa

Mx1
IB: GAPDH 35 kDa
245 kDa
Mock VSV 8 h 135 kDa

i k 100 kDa
Δ D
S- AR

75 kDa
TM

HA-ZNFX1 – FL F1 F2 F3 F4 F5
AV C
M -Δ

FL F1 F2 F3 F4 F5
S

Flag-truncation
S

Flag-MAVS + + + + + + +
AV
AV
M

EV + – – – – – –
M

HA-ZNFX1 + + +
IP: α-HA 75 kDa IP: α-Flag 245 kDa
IB: α-Flag
IB: α-HA
245 kDa
IP: α-HA
135 kDa IP: α-Flag
100 kDa 75 kDa
75 kDa IB: α-Flag
IB: α-HA

IB: α-Flag 75 kDa

IB: α-HA 245 kDa
WCL

245 kDa
135 kDa
WCL

IB: α-HA IB: α-Flag 75 kDa

75 kDa

Fig. 6 | ZNFX1 localises to mitochondria and interacts with MAVS. a, Endogenous ZNFX1 was detected in mitochondrial fractions but not nucleic fractions
of A549 cells by immunoblot analysis. b, Flag-ZNFX1 was expressed in A549 cells for 24 h, then the cells were challenged with VSV. Immunofluorescence
with anti-Flag antibody was used to detect the overexpressed protein (green). Mitochondria were identified by mitochondrial marker (red). Scale bar,
10 μm. c, Mitochondrial fraction isolated from A549 cells was treated with proteinase K (100 ng ml−1) in the absence or presence of 1% TritonX-100
for 15 min on ice, and the reactants were developed by immunoblot analysis. Alkali extraction was carried out using Na2CO3 to treat the mitochondrial
fraction isolated from A549 cells. Both the soluble protein fraction (S) and integral membrane protein fraction (P) were isolated and used for immunoblot
analysis. ‘M’ indicates untreated control mitochondrial sample. d, Co-immunoprecipitation of extracts of 293T cells transfected with Flag-tagged TRIF,
MyD88, MAVS and STING, together with HA-ZNFX1. e–g, Co-immunoprecipitation and immunoblot analysis of A549 cells infected with VSV and
harvested at the indicated time points. WCL, whole-cell lysates. h, Confocal microscopy of 293T cells transfected with Flag-ZNFX1 and GFP-MAVS, with
or without VSV infection. Scale bar, 10 μm. i, Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with Flag-MAVS and deletion
mutants of HA-ZNFX1 or empty vector are shown. j, qRT–PCR analysis of Ifnb1 and ISGs in A549 cells transfected with ZNFX1 and its truncated versions.
k, Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with HA-ZNFX1 and truncation of MAVS. Data are representative of three
independent experiments with similar results.

Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology 1353

Articles
presence of ZNFX1 protein in the mitochondrial fractions isolated
from VSV-infected cells (Fig. 6a, lower panel). Confocal microscopy
confirmed the co-localisation of overexpressed ZNFX1 protein with
mitochondria and enlarged ZNFX1 specks were observed after viral
infection (Fig. 6b). To further explore the sub-mitochondrial locali-
sation of ZNFX1, protease protection assays were performed. As
shown in Fig. 6c, when mitochondria were treated with proteinase K
without Triton X-100, MAVS and most of the ZNFX1 protein were
digested, indicating ZNFX1 as an outer mitochondrial membrane
protein. In contrast, cytochrome c oxidase subunit IV (COX IV), a
mitochondria inner membrane protein, was resistant to proteinase
K digestion. To further characterize the mitochondria association
of ZNFX1, mitochondria was then subjected to alkali extraction.
Supernatant with peripheral membrane proteins and pellet frac-
tions with integral membrane proteins were recovered.
We found that the outer membrane integral protein MAVS was
primarily recovered in the pellet following extraction, but observed
COX IV in both fractions. ZNFX1 was largely recovered in the
supernatant (Fig. 6c), indicating that ZNFX1 localises to mitochon-
dria as a peripheral membrane protein, like SARM1 and ALEX3 in
previous reports37,38,40.
To further characterize how ZNFX1 transduces the antiviral
signals when sensing viral RNA, we examined the interaction of
ZNFX1 with adaptors involved in IFN production, including TRIF,
MyD88, MAVS and STING. Co-immunoprecipitation experi-
ments showed that HA-tagged ZNFX1 was associated with Flag-
tagged MAVS but not with TRIF, MyD88 and STING (Fig. 6d).
Endogenous co-immunoprecipitation experiments further showed
that ZNFX1 and MAVS formed a complex in A549 cells naturally,
and their association was enhanced upon viral infection owing to
the upregulation of ZNFX1 (Fig. 6e–g). Confocal microscopy also
indicated that ZNFX1 co-localised with MAVS (Fig. 6h). Further
co-immunoprecipitation assays using a series of ZNFX1 trunca-
tion mutants showed that full-length ZNFX1 and ARM-containing
mutants, including F3 and F5, could interact with MAVS (Fig. 6i).
Consistent with co-immunoprecipitation assays, overexpressed
full-length and F3 truncation mutant of ZNFX1 resulted in induced
expression of Ifnb1 and ISGs and restricted VSV replication (Fig. 6j
and Extended Data Fig. 4a). Moreover, ZNFX1 could not interact
with truncated MAVS without the transmembrane (TM) domain
(Fig. 6k), and the mitochondrial distribution of ZNFX1 was inde-
pendent of its interaction with MAVS (Extended Data Fig. 4b).
ZNFX1-mediated signalling is independent of RIG-I and MDA5.
To validate the role for ZNFX1 in generating antiviral immune
responses relative to existing antiviral sensors RIG-I and MDA5,
we first performed endogenous co-immunoprecipitation assays and
found that ZNFX1 interacts with neither RIG-I nor MDA5 in rest-
ing or VSV-infected cells. qRT–PCR analyses were performed and
indicated that Znfx1 deficiency did not affect RIG-I-, MDA5- and
MAVS-mediated induction of Ifnb1 expression (Fig. 7a). Moreover,
overexpressed ZNFX1 could intensely induce the mRNA expression
of Ifnb1 and Ifit1 in wild-type and Mda5−/− cells, slightly in Rig-I−/−,
but not in Mavs−/− cells (Fig. 7b). The qRT–PCR results also showed
reduced viral loads of VSV and EMCV in Mda5−/− cells when cells
were transfected with ZNFX1, RIG-I and MDA5 expressing plas-
mids, respectively (Fig. 7c). Likewise, FACS analyses indicated a
reduced percentage of GFP+ cells in VSV-eGFP infected wild-type,
Mda5−/− and Rig-I−/− cells, which were transfected with viral sensors,
including ZNFX1 (shown in Fig. 7d,e). By using RNA sequencing,
we observed that ZNFX1 overexpression enhanced the induction
of IFNs and ISGs in Rig-I−/− cells upon poly(I:C) stimulation (Fig.
7f). We also observed the VSV and poly(I:C) induction of Ifnb1 and
Ifit1 in Znfx1 and Rig-I double knockout cells (DKO) when cells
were transfected with ZNFX1 or RIG-I-overexpressed plasmids,
respectively (Fig. 7g,h). Moreover, siRNA knockdown of ZNFX1
1354 Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology
NATURE CELL BIoLoGy
could further enhance the viral loads of VSV-eGFP in Rig-I−/− cells
(Fig. 7i). Similar results were noted in that the viral loads of VSV-
eGFP in DKO cells were much higher than those in Rig-I−/− cells
(Fig. 7j). All these results suggested that ZNFX1 performs its antivi-
ral roles without depending on RLRs.
Discussion
The innate immune system detects invading viruses mainly by rec-
ognizing the pathogenic nucleic acid through pattern recognition
receptors. Overwhelming evidence has shown that RLRs, includ-
ing RIG-I and MDA5, are perhaps the most important lines of viral
RNA sensing3–6,41. However, expression of RIG-I and MDA5 is rare
in resting cells and becomes detectable until 7 h in viral infection42,43.
Thus, although the antiviral roles of RIG-I and MDA5 have been
well established, how the type I IFN is primed to initiate the early
expression of a series of ISG genes, including RIG-I and MDA5,
remains unanswered.
Here, we have demonstrated that ZNFX1, a member of RNA heli-
case SF1, is an IFN-stimulated gene and can restrict the replication
of RNA viruses. Similar to RIG-I and MDA5, ZNFX1 induces a type
I IFN response by sensing viral RNAs and interacting with MAVS.
However, ZNFX1 differs from RIG-I and MDA5 in several aspects.
First, although RIG-I, MDA5 and ZNFX1 are commonly expressed
ISGs regulated by the Jak-STAT pathway, the constitutive mRNA or
protein levels of ZNFX1 are much higher than those of RIG-I and
MDA5 in various tissues and cell types (Fig. 1i and Extended Data
Figs. 1f, 2f and 5a–c). Meanwhile, the induced expression of ZNFX1
is primed and reaches a peak much earlier than RIG-I upon IFN-α
and IFN-β stimulation (Fig.1i). These differences may indicate the
prime importance of ZNFX1 to monitor viral invasion at an early
stage. Second, unlike RIG-I and MDA5, localised in the cytosol with
cytosol-to-mitochondrial membrane translocation after sensing
viral RNA, ZNFX1 is localised to the outer membrane of mitochon-
dria, which should facilitate its natural interaction with mitochon-
dria-localised MAVS. Although no classical mitochondria targeting
motif can be predicted from ZNFX1, functional studies of the ARM
domain containing proteins ALEX3 and SARM suggest that the
ARM in ZNFX1 may be responsible for its mitochondrial localisa-
tion37,38,44–46. Third, although ZNFX1 can interact with MAVS, the
ZNFX1–MAVS complex is not as efficient as the RIG-I–MAVS
complex in inducing the transcription of Ifnb1 and ISGs. Such a
functional difference may be due to the lack of the caspase recruit-
ment domain (CARD) in ZNFX1. Although ZNFX1 binds to virus
RNA and interacts with MAVS in a fashion independent of RIG-I
and MDA5, the deficiency of ZNFX1 could impair the induced
expression of a series of ISGs, including RIG-I or MDA5, leading
to antiviral defect in the early stage of virus infection47–49. Thus, we
infer that ZNFX1 is probably an early sensor for RNA viruses rather
than a redundant RNA sensor to trigger IFN signalling upon sens-
ing dsRNA. However, the efficient antiviral responses based on the
induction of IFNs are still required for the activation of RLRs, whose
expression could be primed by ZNFX1 in the early antiviral stage.
From this point of view, ZNFX1 might also be regarded as a posi-
tive regulator of RIG-I and MDA5 signalling, leading to an efficient
antiviral signalling network later on (Extended Data Fig. 7).
In addition to the functional differences that have been men-
tioned above, we found that ZNFX1 is highly conserved from fruit
flies to humans (Extended Data Figs. 5d and 6 and Supplementary
Table 1), which is strikingly different from RIG-I, which is lacking
in fruit fly, fugu fish and chicken lineages. In addition to ZNFX1,
another representative of RNA helicase SF1, MOV10, is well con-
served during evolution and shown to be a co-factor to enhance the
nuclear export of viral mRNA34. Thus, the evolutionarily conserved
RNA helicase SF1 rather than RLRs may play more important anti-
viral roles in lower species. The arms race between viruses and the
host may finally result in the recruitment of many RNA or DNA
NATURE CELL BIoLoGy Articles
a WT Znfx1–/– b WT Rig-I –/– Mda5 –/– Mavs–/– WT Rig-I –/– Mda5 –/– Mavs–/–

Rel. mRNA of Ifnb1

Rel. mRNA of Ifnb1
P = 0.1758

Rel. mRNA of Ifit1

1,500 200 P = 0.0007 P = 0.0004 400 P = 0.0294
P = 0.5914 P = 0.0304 P = 0.0004
1,000 150 300 P = 0.0002
P = 0.3851
500 100 200
P = 0.0734
100 50 P = 0.1935 100 P = 0.6618
0 0 0
EV + + + + EV + + + +
1

S
M 5
M I
ZN V

-
FX

A
AV
IG
E

ZNFX1 + + + + ZNFX1 + + + +

D
R
RIG-I + + + + RIG-I + + + +
MDA5 + + + + MDA5 + + + +

c WT d
EV+ RIG-I+ ZNFX1+
80,000 P = 0.0015 Mda5 –/– 40.43% 7.85% 3.40% e
WT Rig-I –/– Mda5 –/–

WT
Rel. mRNA

60,000 P = 8.41 × 10 –5
of VSV

80 P = 0.0097 P = 1.03 × 10–5

40,000 –6
P = 2.93 × 10

(% of total)
VSV-GFP+
60
20,000
EV+ RIG-I+ ZNFX1+ 40
0
59.94% 30.42% 51.41%

Rig-I –/–
EV + + 20
ZNFX1 + +
0
RIG-I + +
EV + + ++ + +
MDA5 + +
ZNFX1 + + +
VSV VSV
EV+ MDA5+ ZNFX1+ RIG-I + +
MDA5 +

Mda5–/–
62.34% 18.37% 13.54%
P = 0.0030 VSV-eGFP VSV-eGFP VSV-eGFP
SSC-A

60,000
P = 0.0005
Rel. mRNA
of EMCV

40,000
VSV-eGFP
20,000

0 g DKO DKO
h DKO DKO
EV + + P = 0.0001
Rel. mRNA of Ifnb1

P = 0.0030 Rel. mRNA of Ifnb1

Rel. mRNA of Ifit1

Rel. mRNA of Ifit1

ZNFX1 + + 15 P = 0.0003 6 100,000 P = 0.0004 1,000

+ + 10,000
RIG-I 100
10 4 1,000
MDA5 + + 100 10
EMCV EMCV 5 2 10
1
1
0 0 0.1 0.1
EV + + EV + + EV + + EV + +
ZNFX1 + + ZNFX1 + + ZNFX1 + + ZNFX1 + +
RIG-I + + RIG-I + +
VSV VSV
Poly(I:C) Poly(I:C)

f i j
Apobec3

WT
Tnfsf10

WT
Parp14
Trim38
Trim25
Trim21

Psmb9

Myd88
Usp18

Dhx58

Rig-I –/–
Tdrd7

Isg20
Ifitm3
Ifitm1

Gbp4
Gbp3
Gbp2

50 50
Hla-e
Hla-b

Cd38
Tap1

P = 0.0026 Rig-I –/–

Ccl5
Ccl2
Ifi35
Ifi16
Nmi
Tlr3

Ifit3
Ifit2
Irf1

P = 0.0051
40 40
(% of total)

(% of total)
VSV-GFP+

VSV-GFP+

DKO
30 30
EV
20 20
Rig-I –/–

ZNFX1 10 10
EV+ 0 0
si 1

1
si si C rl
FX l

si si C rl
FX l
ZN tr

ZN tr

N-STM VSV-eGFP
t

t
C

ZNFX1+
si

Row z-score N-STM VSV-eGFP

–1.5 0 1.5

Fig. 7 | ZNFX1 induces IFN signalling in an RLR-independent manner. a, qRT–PCR analysis of Ifnb1 in wild-type and Znfx1−/−A549 cells transfected
with the indicated expressing plasmids. b, qRT–PCR analysis of Ifnb1 and Ifit1 in wild-type, Rig-I−/−, Mda5−/− or Mavs−/− A549 cells transfected with the
indicated expressing plasmids. c, qRT–PCR analysis of cellular VSV and EMCV loads in wild-type or Mda5−/− A549 cells transfected with the indicated
expressing plasmids for 24 h followed by viral infection for another 12 h. d, FACS analysis of WT, Rig-I−/− and Mda5−/− A549 cells transfected with ZNFX1,
RIG-I or MDA5 expressing plasmids or EV for 24 h followed by VSV-eGFP infection. The experiments were repeated three times, independently, with
similar results obtained. e, Statistical analysis of the data from the multiple repeated FACS experiments in d. f, Heatmap of ISGs expression profiles in
ZNFX1 overexpression in Rig-I−/− cells with or without poly(I:C) stimulation. g,h, Ifnb1 and Ifit1 mRNA transcripts were analysed by qRT–PCR in Rig-I−/− and
Znfx1−/− DKO cells transfected with the indicated expressing plasmids followed by VSV or poly(I:C) stimulation. i, FACS analysis of WT and Rig-I−/− A549
cells transfected with siRNAs of control or Znfx1 for 48 h followed by VSV-eGFP infection. j, FACS analysis of WT, Rig-I−/− and DKO A549 cells followed by
VSV-eGFP infection. For a–c,e,g–j, n = 3 independent experiments. Data are presented as mean ± s.d. P values were calculated using two-tailed unpaired
Student’s t-test.

binding proteins, such as the DExD/H box helicases, to detect the In all, our study here has identified ZNFX1 as a dsRNA sensor
invading viruses in the evolution of vertebrates. As well as acting as that induces the production of IFNs and ISGs in the early stage of
a dsRNA sensor to trigger antiviral responses, ZNFX1 is required viral infection, providing further insights into the existing complex-
for epigenetic inheritance in Caenorhabditis elegans, based on ity of the antiviral immunity mediated by RLRs and MAVS. ZFNX1,
two recent reports50,51. Thus, determining further roles of ZNFX1 as the monitor to sense viral RNA in the early stage of viral infec-
in other physiological or pathological processes still requires tion, may serve as a strategy for early therapeutic intervention in
further studies. viral infections in the future.

Nature Cell Biology | VOL 21 | NOVEMBER 2019 | 1346–1356 | www.nature.com/naturecellbiology 1355

Articles
Online content
Any methods, additional references, Nature Research reporting
summaries, source data, extended data, supplementary informa-
tion, acknowledgements, peer review information; details of author
contributions and competing interests; and statements of code
and data availability are available at https://doi.org/10.1038/s41556-
019-0416-0.
Received: 16 October 2018; Accepted: 26 September 2019;
Published online: 4 November 2019
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NATURE CELL BIoLoGy
Methods
Mice. C57BL/6 Znfx1−/− mice were generated by a CRISPR/Cas9 system from
GemParmatech Co. Exons 3 to 11 were selected as the targeted region. Mice were
kept and bred in pathogen-free conditions. The Institutional Animal Care and
Use Committee of Sun Yat-sen University, Guangzhou, China approved all the
experimental protocols concerning the handling of mice. The study is compliant
with all relevant ethical regulations regarding animals.
Reagents and antibodies. The poly(I:C) (both HMW and LMW) and poly(dA:dT)
were purchased from InvivoGen. Recombinant human IFN-α, recombinant human
IFN-β and all IFN ELISA kits were purchased from R&D Systems. Lipofectamine
2000, Lipofectamine 3000 and Lipofectamine RNAiMAX were purchased from
Invitrogen. Protein G agarose, used for immunoprecipitation, was purchased from
Pierce. Poly(I) and agarose-bond poly(C) were from Sigma (P4154 and P9827).
The following antibodies were used for immunoblot analysis: anti-human ZNFX1
(1:1,000; ab179452, Abcam), anti-mouse ZNFX1 (1:1,000; Dia-An Biotech), anti-
MAVS (1:1,000; ab31334, Abcam), anti-IRF3 (1:1,000; sc-33641, Santa Cruz), anti-
RIG-I (1:1000; D14G6, Cell Signaling), anti-phosphorylated IRF3 (1:1,000; 4D4G,
Cell Signaling), anti-TBK1 (1:1,000; D1B4, Cell Signaling), anti-phosphorylated
TBK1 (1:1,000; D52C2, Cell Signaling), anti-GAPDH (1:10,000; ET1601-4,
HuaBio), anti-β-actin (1:10,000; 60008-1-Ig, Proteintech), anti-lamin B1 (1:2,000;
12987-1-AP, Proteintech), anti-COX-IV (1:3,000; 66110-1-Ig, Proteintech), anti-
HA (1:5,000; clone HA-7, H6533, Sigma), anti-Flag (1:5,000; clone M2, F1804,
Sigma), goat anti-mouse IgG-HRP (1:10,000; HA1006, HuaBio), goat anti-rabbit
IgG-HRP (1:10,000; HA1001, HuaBio).
Cells and pathogens. The A549, MEF, L929 and 293T cell lines were purchased
from ATCC. All these cells were cultured in endotoxin-free DMEM (Gibco),
supplemented with 10% FBS (Gibco) and 1% penicillin–streptomycin (Gibco).
PBMC and mouse peritoneal macrophage were isolated and cultured as described
previously33. VSV (Indiana strain), VSV-eGFP and HSV-1 were from our
laboratory. Human influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) and SeV
were provided by J. Cui (College of Life Sciences, Sun Yat-sen University). H1N1,
SeV, VSV and HSV-1 were titred on Vero cells.
Analogue stimulation and virus infection. Cells were stimulated with poly(I:C)
(3 μg ml−1) and poly(dA:dT) (3 μg ml−1) delivered by polyethylenimine (PEI) for the
indicated hours. Cells were infected at an indicated MOI in serum-free DMEM for
1 h, washed by 1× PBS, and incubated with fresh complete medium for the times
shown in the figures. For in vivo virus infection, six- to eight-week-old mice were
infected with VSV for 24 h by intravenous injection (2 × 107 p.f.u. per g body weight).
RNA interference. Lipofectamine RNAi MAX was used for the transfection
of siRNAs (30 nM) into A549 or L929 cells according to the manufacturer’s
instructions. The sequences of siRNAs with higher silencing efficiency are as follows:
human Znfx1-siRNA: 5′-GGAAUAUCGUGGAGUGUAA dTdT-3′;
human Mavs-siRNA: 5′-CCACCUUGAUGCCUGUGAA dTdT-3′.
These human RNA oligos were synthesized at Guangzhou Ribobio. The mouse
siRNAs were purchased from Thermo Scientific. The target for Znfx1 is ID:509273
and for Mavs is ID:169532.
RNA isolation, qRT–PCR and ELISA. Total RNA was extracted using TRIzol
reagent (Invitrogen) and reversed-transcribed with a PrimeScript RT reagent
kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions. A
Light Cycler480 instrument (using the 384-well module) with RealStar Green Fast
Mixture (GenStar) was used for quantitative real-time PCR analysis. The data were
normalized by the level of β-actin expression in each individual sample. The 2−ΔΔCt
method was used to calculate relative expression changes. The primer sequences
are listed in Supplementary Table 2. The concentrations of IFN-α and IFN-β in
culture supernatants were measured using kits from R&D Systems, according to
the manufacturer’s instructions.
Luciferase reporter gene assay. A549 cells or 293T cells were plated at a density
of 105 cells per well in a 48-well plate. A549 cells in each well were transiently
transfected with 100 ng IFN-β or ISRE luciferase and 5 ng Renilla luciferase
reporter vectors plus ZNFX1 expression vector using Lipofectamine 3000. 293T
cells including wild-type 293T and Znfx1 deficiency 293T cells (Znfx1−/−-293T)
were transfected with reporter vectors using Lipofectamine 2000. At 24 h post
transfection, cells were stimulated with 3 μg ml−1 poly(I:C) or poly(dA:dT) or
infected with the indicated dose of VSV. Cells were harvested 24 h after stimulation
or infection, and luciferase activities were measured with a dual-luciferase reporter
assay system (Promega) according to the manufacturer’s instructions. Data are
normalized for transfection efficiency by dividing Firefly luciferase activity by
Renilla luciferase activity. Each experiment was replicated at least three times.
Library construction for RNA sequencing and sequencing procedures. Total
RNA was isolated using an RNeasy mini kit (Qiagen). RNA integrity was assessed
using an RNA Nano 6000 assay kit of the Agilent Bioanalyzer 2100 system.
Sequencing libraries were generated using an NEBNext UltraTM directional RNA
library prep kit for Illumina (NEB). The poly(A) containing mRNA molecules
Nature Cell Biology | www.nature.com/naturecellbiology
Articles
were purified using poly(T) oligo-attached magnetic beads. Purified libraries
were quantified using a Qubit 2.0 fluorometer (Life Technologies) and validated
using an Agilent 2100 Bioanalyzer to confirm the insert size and to calculate the
mole concentration. Library preparations were sequenced on an Illumina Hiseq
Xten platform and paired-end reads were generated. Library construction and
sequencing were performed at Biomarker Corporation.
Ultraviolet crosslinked RIP assay. 293T cells were transfected with the indicated
Flag-tagged plasmids for 24 h, followed by VSV infection for another 12 h. Cells
were crosslinked with ultraviolet light at 254 nm (300 mJ cm−2), then harvested and
lysed with immunoprecipitation lysis buffer. Flag M2 antibody (Sigma-Aldrich)
was added to the cell lysate and incubated overnight at 4 °C. Protein G beads were
washed and added to antibody–cell lysate mixture and incubated for 2 h at 4 °C.
The bead-bound immunoprecipitates were washed three times with lysis buffer
containing RNase inhibitors. The beads were then split into two samples for protein
or RNA extraction. Proteins were extracted for western blot analysis and the RNA
was extracted using Trizol reagent before real-time PCR analysis for VSV RNA. For
endogenous ZNFX1-RIP analysis, ZNFX1-associated immunoprecipitates were
prepared by incubating IgG or anti-ZNFX1 (2 μg) antibody and protein G beads
with cell lysates of VSV-, EMCV- or SeV-infected A549 cells. RNA was extracted
using Trizol reagent before real-time PCR analysis for viral RNA.
Confocal microscopy. A549 cells were transfected with the expression plasmid
for Flag-tagged ZNFX1. After 24 h, cells were mock or VSV-infected for 8 h and
16 h. The cells were fixed in 4% paraformaldehyde and permeabilized with 0.1%
saponin, then blocked for 30 min with 10% goat serum, incubated overnight at 4 °C
with anti-Flag and mitochondrial marker MitoTracker Red CMXRos (Invitrogen).
Imaging of the cells was carried out using Leica TCS SP5 confocal laser microscopy
under a ×100 oil objective.
Flow cytometry. For gene knockdown, A549 and L929 cells were transiently
transfected with siRNAs for 48 h and then challenged with VSV-eGFP for 6 h
at 2 MOI. For gene overexpression, cells were transfected with the indicated
expression plasmids for 24 h following VSV-eGFP infection at the indicated MOI
for 6 h or 12 h. The cells were washed once in cold PBS and resuspended in cold
staining buffer (PBS with 1% fetal bovine serum). For each condition, 10,000
cells were counted and the followed analyses were performed on a flow cytometer
(FACSCalibur and CytoFLEX).
Mitochondrial fractionation and proteolysis. Mitochondrial fractionation
and proteolysis assays were performed as previously described, with slight
modification41. Briefly, virally infected A549 cells were washed once with cold 1×
PBS (pH 7.2), scraped off the culture plate, and lysed in 800 μl of homogenization
buffer (20 mM HEPES (pH 7.5), 70 mM sucrose and 220 mM mannitol) by 30
strokes in a Dounce homogenizer. The homogenate was centrifuged at 800g
for 5 min to precipitate the nuclei, and the resulting supernatant was further
centrifuged at 10,000g for 10 min (4 °C) to precipitate the crude mitochondrial
fraction. The resulting supernatant was further centrifuged at 100,000g for 30 min
(4 °C) to precipitate light membrane organelles, and the final supernatant was used
as the cytosolic fraction.
For the proteinase K resistance assay, the isolated mitochondria pellet was
washed once with homogenization buffer. Then, the pellet was resuspended in
buffer and treated with proteinase K (100 ng ml−1) in the absence or presence
of 1% Triton X-100 (wt/vol). The proteolytic reaction was performed on ice for
15 min, and the reactants were subjected to western blot analysis with the indicated
antibodies.
Generation of Znfx1−/−, Rig-I−/−, Mda5−/−, Mavs−/− and Ifnar1−/− cells.
Znfx1−/−, Rig-I−/−, Mda5−/−, Mavs−/− and Ifnar1−/− cells were constructed using
the CRISPR/Cas9 gene-editing system. Small guide RNAs (sgRNAs) targeting
the genome sequence of target genes were cloned into PX458 vector. The sgRNA
targeting sequences were 5′-TCCTAACCAACGAGTCTGTT-3′ for ZNFX1,
5′-GCAGGTGCAGAGAAATTGG-3′ for RIG-I, 5′-GGGTGAAAATGTACA
TCCAG-3′ for MDA5, 5′-CCTCTGACCTGGCAGCCCT-3′ for MAVS and
5′-TCATTTACACCATTTCGCAA-3′ for IFNAR1. CRISPR plasmids were
transfected into 293T or A549 cells using Lip2000 and Lip3000, and single-cell
colonies of GFP+ cells were picked and validated at protein expression level by
western blot.
In vitro pulldown and competition. For poly(C) and poly(I:C) pulldown assays,
poly(C)- or poly(I:C)-coated beads were equilibrated in binding buffer and
were combined with an equal volume of whole-cell lysates prepared from 293T
cells transiently transfected with the indicated expression plasmids that were
treated with lysis buffer. The cell extracts were supplemented with protease and
phosphatase inhibitors. The mixtures were incubated with gentle agitation for
2 h at 4 °C. Beads were then washed three times with lysis buffer and analysed by
immunoblotting with anti-HA-HRP.
To generate purified ZNFX1 proteins, 293T cells transiently transfected
with plasmids encoding Flag-ZNFX1 proteins were lysed with lysis buffer. After
Articles
centrifugation, the supernatants were incubated with anti-Flag M2 affinity gel
for 2 h at 4 °C. The beads were washed three times with wash buffer, and the
Flag-ZNFX1 proteins were eluted by competition with Flag peptide. For in vitro
pulldown and competition, purified ZNFX1 proteins were incubated for 4 h with
agarose bead-conjugated poly(I:C) in the absence or presence of poly(I:C) and
other competitors. Centrifugation was followed by the incubation and the beads
were analysed by immunoblotting with anti-HA-HRP.
Statistics and reproducibility. Statistical analyses were carried out using
Microsoft Excel software and GraphPad Prism to assess the differences between
experimental groups. Statistical significance was determined using a two-tailed,
unpaired Student’s t-test with a confidence interval of 95%. P ≤ 0.05 was denoted
as statistically significant. All experiments were performed three or more times
independently under identical or similar conditions.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
Data availability
RNA sequencing data that support the findings of this study have been deposited
in the Gene Expression Omnibus (GEO) under accession code GSE132979. Source
data for Figs. 1–7 and Extended Data Figs. 1–7 have been provided as Statistics
Source Data. All other data supporting the findings of this study are available from
the corresponding author upon reasonable request.
NATURE CELL BIoLoGy
Acknowledgements
This work was supported by the National Natural Science Foundation (NNSF) of China
under grants 31770943, 81430099 and 31900661 and by the Natural Science Foundation
of Guangdong Province of China under grants 2015A030306043, 2018A030313924 and
2018A030313051.
Author contributions
Y.W., S.Y. and A.X. conceived the ideas and designed the experiments. Y.W., X.J., Y.G.,
T.L., M.N. and X.L. performed the experiments. Y.W., S.Y. and X.J. analysed the data. S.Y.,
Y.W., X.J. and S.C. contributed to editing the manuscript. S.Y. and A.X. supervised the
research and wrote the paper. S.Y. and X.J. are joint co first authors.
Competing interests
The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41556-019-0416-0.
Supplementary information is available for this paper at https://doi.org/10.1038/
s41556-019-0416-0.
Correspondence and requests for materials should be addressed to A.X.
Reprints and permissions information is available at www.nature.com/reprints.
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NATURE CELL BIoLoGy Articles

Extended Data Fig. 1 | See next page for caption.

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Articles NATURE CELL BIoLoGy

Extended Data Fig. 1 | Bioinformatics analysis of in vitro transcription-sequencing APA sites (IVT-SAPAS) data of VSV-infected macrophages and
previously collected viral infection microarray data. a, IVT-SAPAS revealed the genes with transcriptional changes in MDMs infected with VSV for 0,
2, 4, 8, 16, 24 hrs. b-c, The percentage of GFP+ cells of FACS analysis of A549 cells transfected with control siRNA or the indicated siRNAs followed by
VSV-eGFP infection for another 6 hrs (n = 3 independent experiments). d, qRT-PCR revealed the mRNA expression of target gene in A549 cells transfected
with indicated siRNAs for 48 hrs (n = 3 independent experiments). e-f, RNA levels of Znfx1 and Rig-I are significantly increased after different virial
infection in different cell types. g, Schematic representation of ZNFX1 promoter containing core region bound by STAT1, STAT2, IRF1 and IRF9. For e, n = 3
independent experiments. Data in f, n = 4 wells for SeV infected epithelial cells and n = 6 wells for uninfected cells; n = 5 samples for IVA infected pDCs;
n = 4 independent experiments for SeV infected monocytoid cells; n = 2 independent experiments for SeV or HIV infected Macrophages or mDCs. All data
are shown as the mean ± s.d. Statistical differences were detected using two-tailed unpaired Student’s t-tests.

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NATURE CELL BIoLoGy Articles

Extended Data Fig. 2 | ZNFX1 deficiency impairs antiviral immune response in vitro and in vivo. a, Quantitative RT-PCR (qRT-PCR) analysis of Znfx1
mRNA expression in A549 and L929 cells transfected with control siRNA or ZNFX1 siRNA 1#, 2# or 3# for 48 hrs (n = 3 independent experiments).
b, Western blot analysis of ZNFX1 protein expression in A549 cells transfected with control siRNA or human ZNFX1 siRNA 1# for 48 hrs. c, ELISA of IFN-α
or IFN-β production in cell supernatants from A549 cells with target gene knockdown for 48 hrs followed by VSV infection or poly I:C stimulation for
another 12 hrs (n = 4 independent experiments). d, qRT-PCR analysis of VSV mRNA expression (left panel) and plaque assay analysis of VSV titer (right
panel) of A549 cells transfected with RIG-I, ZNFX1 expressing plasmids or empty vector plasmid for 24 hrs and then infected with VSV at an MOI of 2 for
16 hrs (n = 3 independent experiments). e, FACS analysis of Znfx1+/+ and Znfx1-/- 293T cells followed by VSV-eGFP infection at 0.5 MOI for the indicated
time points (n = 3 independent experiments). f, Znfx1-/- 293T and A549 clones were generated by the CRISPR-Cas9 method. Deficiency of target genes in
the KO clones were confirmed by immunoblotting analysis. g, qRT-PCR analysis of viral mRNA transcripts in VSV, EMCV, H1N1 and HSV-1 infected A549
cells with control siRNA (si Control) or Znfx1-specific siRNA (si ZNFX1) (n = 3 independent experiments). h, ELISA of IFN-α in supernatants of BMDMs
from WT and Znfx1-/- mice infected with VSV or HSV-1 for 16 hrs (n = 5 independent experiments). All data are shown as the mean ± s.d. P values were
calculated using two-tailed unpaired Student’s t-test. For b, f, the experiments were repeated three times, independently, with similar results obtained.

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Articles NATURE CELL BIoLoGy

Extended Data Fig. 3 | ZNFX1 positively regulates IFN-β signaling. a, Illustration of the CRISPR-Cas9 strategy to generate Znfx1-deficient mice and primer
design used in (b). b, Genotyping of the ZNFX1 mutant pups. c, Immunoblot analysis of ZNFX1 protein levels in Znfx1+/+ and Znfx1-/- MEFs cells. d, qRT-
PCR analysis of Ifnb1 and ISGs mRNA levels in A549 cells transfected with siControl (si Ctrl) or si ZNFX1 for 48 hrs and then infected with VSV for the
indicated time points (n = 3 independent experiments). e, qRT-PCR analysis of Ifnb1 and ISGs mRNA expression in Znfx1+/+ and Znfx1-/- 293T cells followed
by VSV infected with increasing MOI (0.5 and 1) for 0, 8 and 16 hrs (n = 3 independent experiments). For b, c, the experiments were repeated three times,
independently, with similar results obtained. Data in d, e are the mean ± s.d. P values were calculated using two-tailed unpaired Student’s t-test.

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NATURE CELL BIoLoGy Articles

Extended Data Fig. 4 | ZNFX1 localizes to mitochondria and interacts with MAVS. a, FACS analysis of Znfx1-/- A549 cells transfected with ZNFX1 and
its mutants expressing plasmids or empty vector (EV) for 24 hrs followed by VSV-eGFP infection at an MOI of 2 for 6 hrs. b, Endogenous level of ZNFX1
protein in mitochondrial fractions in WT and Mavs-/- 293T with or without VSV infection at 1 MOI for 6 hrs. COX-IV was used as the loading control. Data
are representative of at least three independent experiments.

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Articles NATURE CELL BIoLoGy

Extended Data Fig. 5 | The expression of Znfx1 in different tissues and cell types, and the phylogenetic tree of Znfx1. a-c, The expression of Znfx1, Rig-I
and Mda5 in different tissues and cell types as per BioGPS. d, Phylogenetic tree of ZNFX1 and RIG-I using an amino acid sequence alignment among
different species.

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NATURE CELL BIoLoGy Articles

Extended Data Fig. 6 | Alignment of ZNFX1 amino acid sequences in human, mouse, rat and zebrafish. Shading indicates sequence conservation, with
darker gray indicating a higher degree of conservation.

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Articles NATURE CELL BIoLoGy

Extended Data Fig. 7 | Work model of mitochondria-localized ZNFX1 functions as a dsRNA sensor to initiate antiviral responses through MAVS. Upon
RNA virus infection, ZNFX1 induces type I interferon response by interacting with MAVS in the early stage, thus primes the expression of a number of
ISGs, including RIG-I and MDA5. The induced sensors further enhance the antiviral immune response by amplifying ISGs expression.

Nature Cell Biology | www.nature.com/naturecellbiology

 nature research | reporting summary
Corresponding author(s): Anlong Xu
Last updated by author(s): 28th August 19

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Antibodies
Antibodies used anti-ZNFX1 (IB:1:1000; ab179452, Abcam),
anti-MAVS (IB:1:1,000; IP:1:100, ab31334, Abcam),
anti-IRF3 (IB:1:1,000, sc-33641, SL-12, Santa Cruz),
anti-RIG-I (IB:1:1000, D14G6, #3743, Cell Signaling),
anti-MDA5 (IB:1:1000, D74E4, #5321, Cell Signaling)
anti-phosphorylated IRF3 (IB:1:1,000; 4D4G; #4947, Cell Signaling),
anti-TBK1 (IB:1:1000; D1B4; #3504, Cell Signaling),
anti-phosphorylated TBK1 (IB:1:1000; D52C2, #5483, Cell Signaling),
anti-GAPDH (IB:1:10,000; ET1601-4; HuaBio),
anti-b-actin (IB:1:10,000; 60008-1-Ig; Proteintech),
anti-Lamin B1 (IB:1:2000; 12987-1-AP; Proteintech),
anti-COX-IV (IB:1:3000; 66110-1-Ig; Proteintech),
anti-HA (IB:1: 5,000; clone HA-7, H6533; Sigma),
October 2018

anti-Flag (IB:1:5000; clone M2, F1804, Sigma),

Goat anti-mouse IgG-HRP (IB:1:10000; HA1006, HuaBio),
Goat anti-Rabbit IgG-HRP (IB:1:10000; HA1001, HuaBio),
anti-mouse ZNFX1 protein was prepared by Dia-An Biotech, Inc, in Wuhan, China (IP:1:100; IB:1:1000).

Validation All antibodies used in this study were obtained from commercial sources and validated according to manufacturers's instruction.

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Methodology
Sample preparation For gene knockdown, knockout or overexpression, A549 and L929 cells were transiently transfected with siRNAs or expression
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Instrument Cytoflex (Beckmam Coulter) system.

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