Article

Leaked genomic and mitochondrial DNA contribute

to the host response to noroviruses in a STING-
dependent manner
Graphical abstract Authors
Aminu S. Jahun, Frederic Sorgeloos,
Yasmin Chaudhry, ..., Iliana Georgana,
Rhys Izuagbe, Ian G. Goodfellow

Correspondence
asj40@cam.ac.uk (A.S.J.),
ig299@cam.ac.uk (I.G.G.)

In brief
Jahun et al. show leakage of nuclear and
mitochondrial DNA into the cytosol in
norovirus-infected cells, which activates
the cGAS-STING pathway and
contributes to the overall interferon
response. This leakage of DNA appears to
be driven by the viral NS4 protein, in a
manner dependent on virus replication.

Highlights
d cGAS, IFI16, and STING are required for a robust IFN
response against noroviruses

d Nuclear and mitochondrial DNA accumulate in the cytosol of

norovirus-infected cells

d Accumulation of cytosolic DNA depends on virus replication

d Viral NS4 protein mediates leakage of DNA into the cytosol of

infected cells

Jahun et al., 2023, Cell Reports 42, 112179

March 28, 2023 ª 2023 The Author(s).
https://doi.org/10.1016/j.celrep.2023.112179 ll
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Article
Leaked genomic and mitochondrial DNA contribute
to the host response to noroviruses
in a STING-dependent manner
Aminu S. Jahun,1,* Frederic Sorgeloos,1,2 Yasmin Chaudhry,1 Sabastine E. Arthur,1,3 Myra Hosmillo,1 Iliana Georgana,1
Rhys Izuagbe,1 and Ian G. Goodfellow1,4,*
1Division of Virology, Department of Pathology, University of Cambridge, Addenbrooke’s Hospital Level 5, Hills Road, Cambridge CB2 0QQ,

UK
2Université catholique de Louvain, de Duve Institute, MIPA-VIRO 74-49, 74 Avenue Hippocrate, B-1200 Brussels, Belgium
3Present address: Department of Medical Microbiology, Amsterdam University Medical Center, University of Amsterdam, and Centre for

Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, 3721, Bilthoven,
the Netherlands
4Lead contact

*Correspondence: asj40@cam.ac.uk (A.S.J.), ig299@cam.ac.uk (I.G.G.)

https://doi.org/10.1016/j.celrep.2023.112179

SUMMARY

The cGAS-STING pathway is central to the interferon response against DNA viruses. However, recent studies
are increasingly demonstrating its role in the restriction of some RNA viruses. Here, we show that the cGAS-
STING pathway also contributes to the interferon response against noroviruses, currently the commonest
causes of infectious gastroenteritis worldwide. We show a significant reduction in interferon-b induction
and a corresponding increase in viral replication in norovirus-infected cells after deletion of STING, cGAS,
or IFI16. Further, we find that immunostimulatory host genome-derived DNA and mitochondrial DNA accu-
mulate in the cytosol of norovirus-infected cells. Lastly, overexpression of the viral NS4 protein is sufficient
to drive the accumulation of cytosolic DNA. Together, our data find a role for cGAS, IFI16, and STING in the
restriction of noroviruses and show the utility of host genomic DNA as a damage-associated molecular
pattern in cells infected with an RNA virus.
INTRODUCTION NLRP6 depletion on virus replication in vivo is only marginal.8 In
addition, the depletion of signal transducer and activator of tran-
Noroviruses are positive-sense single-stranded RNA viruses, scription 1 (STAT1), a central mediator of signaling downstream
with approximately 7.4-kb genomes comprising three to four of IFN receptors, leads to profoundly higher viral titers than those
open reading frames and a poly(A) tail.1 The human noroviruses obtained after MDA5 depletion alone, both in vivo6,9 and
(HuNoVs), against which there are no approved vaccines or in vitro.6,10 Together these suggest the presence of other recep-
treatment, are the commonest causes of infectious gastroenter- tors or pathways that contribute to innate immune responses
itis worldwide, with up to 677 million cases and more than against noroviruses.11,12
200,000 deaths every year in children under 5.2–4 The murine While the cyclic guanosine monophosphate-AMP synthase
norovirus (MNV) is broadly used as a surrogate model for study- (cGAS)-stimulator of IFN genes (STING) pathway is mostly associ-
ing the biology of noroviruses because of the availability of a ated with restriction of DNA viruses,5 there have been reports of its
reverse genetics system and a robust animal model, as well as role in the restriction of replication of RNA viruses, in both IFN-
an efficient virus culture system using widely available cell lines dependent and -independent manners.13–17 This is thought to be
and primary cells.1 mediated by the presence of leaked mitochondrial DNA (mtDNA)
Viruses and other pathogens are detected by a myriad of re- in the cytosol resulting from viroporin-mediated calcium imbal-
ceptors whose activation initiates signaling cascades that trigger ances,14 infection-induced mitochondrial damage,13,17 or via an
the release of interferons (IFNs).5 Using MNV as a model, the re- as yet undefined mechanism downstream of IL-1b receptor
ceptors melanoma differentiation-associated protein 5 (MDA5) signaling.18 This leaked mtDNA acts as a damage-associated mo-
and NOD-like receptor family pyrin domain-containing 6 lecular pattern (DAMP), activating host responses downstream of
(NLRP6) have been previously demonstrated to play important STING. Thus far, there are no reports of genomic DNA acting as
roles in initiating IFN responses after infection with noroviruses, DAMPs in the same manner, although previous studies have
both in vivo and in primary cells.6–8 The distribution of these re- demonstrated STING activation after genomic DNA leakage into
ceptors is complementary and varies with cell type, such that the cytosol in tumor cells,19,20 after extensive DNA damage from
MDA5 is largely expressed in myeloid cells, while NLRP6 is pre- irradiation21 or from exposure to some anticancer agents.22
dominantly expressed in epithelial cells.8 However, the effect of Indeed, STING-dependent IFN responses to leaked genomic

Cell Reports 42, 112179, March 28, 2023 ª 2023 The Author(s). 1
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Figure 1. MNV induces IFNs only in STING-competent cells

(A) Cells were infected at the indicated MOIs with either wild-type MNV1 or the VF1 M1 mutant, harvested 10 h after infection and subjected to RT-qPCR. Data are
representative of two independent experiments done in triplicates, and is shown relative to mock-infected cells, and normalized to human b-actin.
(B) Cells were infected at an MOI of 10 with wild-type MNV1, harvested 10 h after infection and assessed by western blotting for the indicated proteins.
(C) Cells were infected at the indicated MOIs with wild-type MNV1, harvested 10 h after infection and infectious viral titers were determined using TCID50. Data
are representative of two independent experiments, each done in triplicates.
(D and E) HeLa-CD300lf cells were infected with wild-type MNV1 at an MOI of 5, and seeded on coverslips. 10 h after infection, the cells were fixed, permeabilized,
and stained for pSTING (red), dsRNA (green), and nuclei (blue), and images were acquired using a confocal microscope. Data in (E) are representative of a
minimum of 100 random cells counted from two independent experiments. All error bars represent standard error of the mean. See also Figure S1.

DNA are thought to significantly contribute to the chemothera-
peutic activities of certain anticancer drugs.23–25
In the current study, we observed a considerable attenuation of
IFN responses and a commensurate increase in viral replication
in norovirus-infected primary cells and cell lines after treatment
with small molecule inhibitors of STING. We also show a
decrease in the induction of IFNs and IFN-stimulated genes
(ISGs) in norovirus-infected STING
/
cells, as well as cGAS
/

and IFI16
/
cells. Mechanistically, we demonstrate a substantial
accumulation of nuclear DNA and, to a lesser extent, mtDNA in
the cytosol of infected cells, likely mediated by the viral NS4 pro-
tein. In summary, we show a host-pathogen dynamic whereby
genomic DNA acts as a DAMP in cells infected with an RNA virus.
RESULTS
MNV induces IFNs only in STING-competent cells
The MNV VF1 protein counteracts induction of type I IFNs through
an as yet unclear mechanism.26,27 A common and significant
challenge in studying the functions of MNV proteins, including
VF1, is that primary murine macrophages and MNV-permissive
cell lines are often difficult to transfect,28,29 and the introduction
of foreign DNA itself frequently leads to the induction of IFNs.
To circumvent these limitations, we transduced two easy-to-
transfect cell lines, HeLa and HEK293T, with the MNV receptor
CD300lf to make them permissive to MNV infection.30,31 To
examine whether VF1 inhibits IFN induction in human cells, the
CD300lf-expressing HeLa and HEK293T cells were infected
with wild-type MNV1 or the previously described VF1-negative
mutant M1 in which a stop codon was introduced at position 17
of the VF1 gene without affecting the underlying VP1 sequence.26
As seen previously in RAW264.7 cells,26 HeLa-CD300lf cells in-
fected with MNV1 had a substantial increase in IFN-b, with the
M1 virus inducing higher levels of IFN-b compared with cells
that were infected with wild-type MNV1 (Figure 1A). By contrast,
there was little or no IFN-b induction in HEK293T-CD300lf cells in-
fected with either the wild-type virus or M1, with no difference
seen in IFN induction between cells infected with the wild-type
MNV1 compared with those infected with the M1 mutant (Fig-
ure 1A). These findings suggest that a factor or pathway present
in HeLa and RAW264.7 cells, but absent in HEK293T, is required
for both a robust induction of IFN in MNV-infected cells, as well as

2 Cell Reports 42, 112179, March 28, 2023


Article
the phenotypic differences observed between cells infected with
the wild-type and M1 viruses.
To further examine IFN induction in HeLa-CD300lf and
HEK293T-CD300lf cells after infection with MNV, the cells
were infected with wild-type MNV1 at a high multiplicity of
infection (MOI), harvested 10 h after infection and the lysates
assessed using western blotting. As shown in Figure 1B (right),
there was no difference in the levels of phospho-IRF3 and
phospho-STAT1 between the mock-infected cells and cells in-
fected with MNV1 in HEK293T-CD300lf cells. This contrasts
with infection in HeLa-CD300lf cells (Figure 1B, left), where a
significant increase is seen in the levels of both phospho-
IRF3 and phospho-STAT1 after infection with MNV1. We also
observed profoundly higher levels in viral titers from the
HEK293T-CD300lf cells compared with HeLa-CD300lf cells
(Figure 1C). The HEK293T-CD300lf cells have a functional
RNA-sensing pathway; they are able to phosphorylate STAT1
in response to poly(I:C) transfection (Figure S1A). Both being
epithelial-like cell lines, HeLa and HEK293T cells likely express
the same repertoire of components of the IFN response
pathway, with the exception of the adapter protein STING
that is only marginally expressed in HEK293T cells, compared
with HeLa cells (Burdette et al.32) (Figure S1B). Taken together,
these data demonstrate an attenuation of the IFN response to
MNV1 infection in HEK293T-CD300lf cells, compared with
HeLa-CD300lf and RAW264.7 cells, putatively suggesting a
role for STING, or other factors non-functional in HEK293T-
CD300lf cells, in the IFN response against noroviruses.
Additionally, we observed phosphorylation of STING in approx-
imately one-third of HeLa-CD300lf cells infected with MNV1
(Figures 1D and 1E), further supporting a role for STING in
the host response to noroviruses.
STING inhibition or deletion attenuates the antiviral
response against MNV
To explore the role of STING in the antiviral response against
MNV, we made use of the recently described covalent small
molecule inhibitors of STING, namely, C-176 and H-151.33 As
shown in Figure 2A, both molecules were able to inhibit induc-
tion of IFN-b after transfection of synthetic double-stranded
DNA (poly[dA:dT]) in RAW264.7 cells, but not RNA (poly[I:C]),
consistent with previously published results.33 To determine
whether STING is required for the induction of IFNs after infec-
tion with MNV, RAW264.7 cells were pre-treated with dimethyl
sulfoxide (DMSO) or titrated doses of C-176 or H-151, and in-
fected with wild-type MNV1 at a high MOI. The cells were
harvested 9 h after infection and subjected to RT-qPCR. As
shown in Figure 2B, there was a significant dose-dependent
decrease in IFN-b induction in cells treated with either C-176
or H-151, compared with DMSO controls. These data suggest
that STING is required for a robust induction of IFNs in
RAW264.7 cells infected with MNV1.
To examine the role of STING in primary macrophages, bone
marrow cells from C57BL/6 mice were differentiated into bone
marrow-derived macrophages (BMDMs), pre-treated with either
DMSO or titrated doses of C-176 or H-151 and subsequently in-
fected with MNV1 at a high MOI. The cells were harvested 12 h
after infection and subjected to RT-qPCR. As shown in Figure 2C,
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OPEN ACCESS
and consistent with data from assays in RAW264.7 cells, there
was a significant dose-dependent decrease in IFN-b induction
in MNV-infected BMDMs after treatment with the small molecule
inhibitors of STING. To determine the role of STING in restricting
MNV1 replication, BMDMs were pre-treated with DMSO or
titrated doses of C-176 or H-151, and infected with MNV1. The
samples were harvested at different time points after infection
and infectious viral titers were determined using TCID50. As
shown in Figure 2D, there was a significant dose-dependent
increase in viral titers after treatment with STING inhibitors.
Altogether, these data indicate that STING plays an important
role in the antiviral responses to MNV1 in both primary macro-
phages and cell lines.
To confirm the role of STING in the IFN responses against
noroviruses, we used the CRISPR-Cas9 system34 to generate
two clones of STING knock-out RAW264.7 cells (KO-1 and
KO-2, Figure 2E) using a previously published single guide
RNA.35 While the baseline levels of IFN-b were decreased in
the STING-KO clones, both induced IFN-b at similar levels to
the wild-type cells after transfection of poly(I:C), but showed
a significantly impaired response to transfected poly(dA:dT),
as expected (Figure S2A), and in agreement with previously
published reports.16 To determine the effect of STING depletion
on IFN-b response to MNV, we performed RNA sequencing on
wild-type RAW264.7 cells and the two STING-KO clones (KO-1
and KO-2) infected with MNV1 at an MOI of 5 and harvested at
9 h after infection. We observed a considerable decrease in
both upregulated and downregulated genes when MNV1-in-
fected STING-KO cells were compared with mock-infected
cells versus when MNV1-infected wild-type cells were com-
pared with mock-infected cells, and when MNV1-infected
STING-KO cells were compared with MNV1-infected wild-
type cells (Figures S2B and S2C, and Table S5). Compared
with infected wild-type cells, both infected KO-1 and KO-2 cells
showed downregulation of type I IFN genes and several ISGs,
including viperin, IFIT1, ISG15, MX1, IFI44, and IFI44l, (Figures
2F and 2G). Assessment of the 215 commonly differentially ex-
pressed genes between infected KO-1 and KO-2 cells when
each was compared with infected wild-type cells showed a
high correlation, with a Pearson correlation coefficient of 0.8
(Figures S2D and S2E). Transcription factor enrichment anal-
ysis indicated a significant enrichment of genes that are pre-
dicted targets of STAT1 (Figure S2F), and gene ontology and
reactome analyses of these genes showed a significant enrich-
ment of genes involved in host antiviral responses, including
IFN responses (Figures S2G and S2H).
Using RT-qPCR, we observed a similar decrease in IFN-b and
ISGs in both KO-1 and KO-2 after infection with MNV1 (Fig-
ure S3A), which was partially rescued after transduction with a
CRISPR-resistant mouse STING (Figures S3B–S3D). In addition,
there was a significant increase in viral titers in both KO-1 and
KO-2 cells (Figure 2H), consistent with data obtained from
RAW264.7 cells transduced with short hairpin RNA targeting
STING (Figures S3E–S3H). Similar to HeLa-CD300lf cells, we
observed phosphorylation of STING in approximately 25% of
RAW264.7 cells infected with MNV1 (Figures 2I and 2J). Alto-
gether, these data confirm a role for STING in the antiviral
responses to MNV1 infection.
Cell Reports 42, 112179, March 28, 2023 3
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representative of two independent experiments, each done in triplicate.
(I and J) RAW264.7 cells were infected with wild-type MNV1 at an MOI of 5, and seeded on coverslips. At 10 h after infection, the cells were fixed, permeabilized,
and stained for pSTING (red), dsRNA (green), and nuclei (blue), and images were acquired using a confocal microscope. Data in (I) represent a minimum of 100
random cells counted from two independent experiments. All error bars represent standard error of the mean. See also Figures S2 and S3.
STING inhibition enhances replication of HuNoVs in cell
lines and biopsy-derived organoids
Next, we wanted to see if there is a similar role for STING in the
IFN responses against HuNoVs. For this, we made use of the
recently described HGT-NV cells,36 a human gastric tumor cell
line stably harboring a GI.1 HuNoV replicon. The cells were
treated with DMSO or titrated doses of H-151. As shown in
Figures 3A and 3B, inhibition of STING led to a significant in-
4 Cell Reports 42, 112179, March 28, 2023
Article
Figure 2. STING inhibition and deletion im-
pairs IFN induction in MNV-infected cell
lines and primary cells
(A) RAW264.7 cells pre-treated with DMSO,
0.5 mM C-176 or 0.5 mM H-151 for 2 h, were
transfected with 1 mg poly(I:C) or poly(dA:dT). The
cells were harvested after 2 h and subjected to RT-
qPCR. Data are representative of experiments
done in triplicates, and is shown relative to mock-
transfected cells, and normalized to mouse
Gapdh.
(B) RAW264.7 cells pre-treated with DMSO or the
indicated amounts of C-176 or H-151 for 2 h were
infected with wild-type MNV1 at an MOI of 10. The
cells were harvested 9 h after infection and sub-
jected to RT-qPCR. Data are representative of two
independent experiments done in triplicates, and
is shown relative to mock-infected cells, and
normalized to mouse Gapdh.
(C) BMDM cells pre-treated with DMSO or the
indicated amounts of C-176 or H-151 for 2 h were
infected with wild-type MNV1 at an MOI of 10. The
cells were harvested 12 h after infection and sub-
jected to RT-qPCR. Data are representative of two
independent experiments done in triplicates, and
is shown relative to mock-infected cells, and
normalized to mouse Gapdh.
(D)BMDM cells pre-treated with DMSO or the
indicated amounts of C-176 (left) or H-151 (right)
for 2 h were infected with wild-type MNV1 at an
MOI of 10. The samples were harvested at different
time points after infection and infectious viral titers
were determined using TCID50. Data are repre-
sentative of two independent experiments, each
done in triplicates.
(E) Wild-type and two clones of STING-KO (KO-1
and KO-2) RAW264.7 cells were assessed by
western blotting for the indicated proteins.
(F and G) Wild-type and two clones of STING-KO
(KO-1 and KO-2) RAW264.7 cells were infected
with wild-type MNV1 at an MOI of 5 and harvested
at 9 h after infection. Samples were assessed by
RNA sequencing. Data are presented as volcano
plots of differentially expressed genes of indicated
comparisons, with genes colored green or red
indicating those with adjusted p value of less than
0.05 and a fold change of 2 or more. Data repre-
sent a single experiment done in triplicate.
(H) Wild-type and STING-KO RAW264.7 cells,
were infected with wild-type MNV1 at an MOI of 5
and harvested at 9 h after infection. Infectious viral
titers were determined using TCID50. Data are
crease in the HuNoV genomes 24 h after treatment (Figure 3A),
and viral proteins at 72 h after treatment (Figure 3B), in a dose-
dependent manner. We also saw a marked increase in GII.4
and GII.3 HuNoV genomes in three previously described bi-
opsy-derived human intestinal organoids37,38 spanning the three
major regions of the small intestine (Figures 3C–3E). Taken
together, these results suggest that STING contributes to the re-
striction of both human and mouse noroviruses during

Article
replication, although we failed to observe its phosphorylation in
the replicon-containing cells (Figure S4).
Both cGAS and IFI16 contribute to IFN responses in
norovirus-infected cells
Next, the upstream receptor that mediates STING activation in
MNV-infected cells was examined. Activation of either cGAS or
IFI16 leads to the activation of STING and eventually the induction
of IFNs.5 To explore the roles of these receptors in norovirus-in-
fected cells, we used RAW264.7 cells stably expressing a secreted
luciferase under the control of the ISG54 (ISRE) promoter (RAW-
Lucia ISG cells), with different cell lines lacking proteins involved
in the detection of either cytosolic RNA or DNA (Figures 4A and
4B). Wild type, cGAS
/
or IFI16
/
(p204
/
) cells were infected
with MNV1 at an MOI of 5. As controls, MAVS
/
, STING
/
, and
MDA5
/
were also infected at the same MOI. The supernatants
were harvested at 18 h after infection and analyzed for biolumines-
cence. As shown in Figure 4C, there was a significant decrease in
IFN-b induction in both cGAS
/
and IFI16
/
, compared with wild-
type cells, and comparable with the decrease seen in MAVS
/
,
STING
/
, and MDA5
/
cells. This was also true for IFN-b
mRNA levels (Figure 4D). Using confocal microscopy, we observed
phosphorylation of STING in wild-type RAW-Lucia ISG cells, but
not in cGAS
/
and IFI16
/
cells (Figure 4E), suggesting that
both cGAS and IFI16 are required for STING activation and robust
induction in IFN-b in MNV-infected cells.
While there was no difference in infectious viral titers in
cGAS
/
and IFI16
/
RAW-lucia ISG cells infected with MNV1
compared with wild-type cells (Figure 4F), there was approxi-
mately 7 and 9 times more viral genome copies in cGAS
/
and
IFI16
/
cells, respectively (Figure 4G). This discrepancy
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Figure 3. STING inhibition enhances repli-
cation of HuNoVs in replicon-containing
cell lines and biopsy-derived organoids
(A) Pre-seeded HGT-NV cells were treated with
DMSO or indicated doses of H-151. The cells were
harvested 24 h after treatment and subjected to
RT-qPCR. Data are representative of two inde-
pendent experiments done in triplicates, and is
shown relative to DMSO-treated cells, and
normalized to human b-actin.
(B) Pre-seeded HGT-NV cells were treated with
DMSO, H-151 (+ = 0.5 mM, ++ = 5 mM), or 20 -C-
methylcytidine (2CMC, 200 mM). The cells were
harvested 72 h after treatment and assessed by
western blotting for the indicated proteins.
(C–E) Monolayers generated from the Ti365 (C),
D196 (D), and J2 (E) human intestinal organoids
were infected with GII.4, GII.3, and GII.3 HuNoVs,
respectively, after pre-treatment with either DMSO
or H151. The cells were harvested 48 h after
infection and viral genomes were assessed by
qPCR. Data are representative of two independent
experiments done in duplicate and are shown
relative to day 0 DMSO-treated cells. D0 = day 0,
D2 = day 2. All error bars represent standard error
of the mean. See also Figure S4.
between infectious viral titers and genome copy numbers, which
is also true for MAVS
/
, STING
/
, and MDA5
/
cells as well as
cells treated with the JAK1/JAK2 inhibitor ruxolitinib (Figures 4F
and 4G), is not explained by increased cell death; no significant
decrease in cell viability was observed in any of the KO cells at
the time of harvest (Figure 4H). This suggests that, in wild-type
cells, the encapsidation step and not genome replication is the
limiting factor for infectious virus production in this in vitro model
of norovirus infection.
Aberrant cytosolic DNA from norovirus-infected cells
induces an IFN response in a STING-dependent manner
Mitochondrial DNA has been demonstrated to accumulate in the
cytosol of host cells after infection by some RNA viruses,
including Dengue virus,13 Chikungunya virus,15 influenza A
virus,14 and encephalomyocarditis virus (EMCV).14 This aberrant
presence of DNA in the cytosol then leads to activation of the
cGAS-STING pathway and the ensuing IFN response. To deter-
mine if mtDNA accumulates in the cytosol of norovirus-infected
cells, RAW264.7 cells were infected at an MOI of 0.5 or 5, and
harvested at 12 h after infection. The cells were then lysed in a
digitonin buffer and fractionated (Figures S5A and 5A). DNA
was extracted from the cytosolic fractions and the presence of
mtDNA was determined by qPCR using two previously described
primer sets designed to detect different regions of the
mitochondrially encoded cytochrome c oxidase I (Cox1)
gene.14,39 As shown in Figures 5B and S5B, there was a moderate
accumulation of mtDNA in the cytosol of infected cells, with
higher levels observed with an increased MOI. Interestingly, we
also observed a sizable dose-dependent increase in genome-
derived Gapdh (indicated as nuclear-DNA1) and Hprt (indicated
Cell Reports 42, 112179, March 28, 2023 5
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Figure 4. Both cGAS and IFI16 contribute to IFN responses in norovirus-infected cells
(A) Schematic representation of the IFN induction pathway downstream of cytosolic DNA and RNA sensing.
(B) Wild-type (WT) RAW-Lucia ISG cells or those with the indicated KOs were transfected with 1 mg/mL Poly(I:C), or with 1 mg/mL Poly(dA:dT). The supernatants
were harvested 18 h after infection and analyzed for Lucia luciferase levels on a luminometer. Data are presented as fold increase relative to mock.
(C) Cells were infected with MNV1 at an MOI of 5. The supernatants were harvested 18 h after infection and analyzed for Lucia luciferase levels on a luminometer.
Data are representative of two independent experiments done in triplicates and are presented as fold increase relative to mock.
(D) Cells were infected with WT MNV1 at an MOI of 5. The cells were harvested 9 h after infection and assessed by RT-qPCR. Data are representative of two
independent experiments done in triplicates and shown relative to control and normalized to Gapdh.
(E) Cells were infected with WT MNV1 at an MOI of 5 and seeded on coverslips. At 10 h after infection, the cells were fixed, permeabilized, and stained for pSTING
(red), dsRNA (green), and nuclei (blue), and images were acquired using a confocal microscope.
(F–H) Cells were infected with WT MNV1 at an MOI of 5, with or without treatment with 10 mM ruxolitinib (Rux). The cells were harvested at 9 h after infection and
infectious viral titers were determined using TCID50 (F), genome copies were determined using RT-qPCR (G), or cell viability was determined using the CellTiter-
Blue assay (H). Data are representative of two independent experiments, each done in triplicate. All error bars represent standard error of the mean.

as nuclear-DNA2) in the cytosol of infected cells (Figures 5C and
S5B). This leakage of mitochondrial and genomic DNA into the
cytosol depends on virus replication; it was significantly
decreased in the presence of 20 -C-methylcytidine (2CMC)
(Figures 5D, 5E, and S5C), a known inhibitor of norovirus replica-
tion.40,41 Additionally, we observed higher levels of cytosolic DNA
in GI.1 HuNoV replicon-containing HGT-NV cells, compared with
the parental cell line, which was significantly reduced after treat-
ment with 2CMC (Figures S5D and S5E). Taken together, these
data indicate that both mitochondrial and genomic DNA are
released into the cytosol of norovirus-infected cells.
To determine if this cytosolic DNA is immunostimulatory in the
context of norovirus infection, we treated RAW264.7 cells with
low doses of ethidium bromide (EtBr) for 48 h to deplete mitochon-
drial and other extra-chromosomal DNA, as previously des-
cribed.42,43 This decreased mtDNA by almost 90% (Figure 5F),

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Figure 5. Cytosolic DNA accumulates in norovirus-infected cells, and induces an IFN response in a STING-dependent manner
(A–C) RAW264.7 cells were infected with MNV1 at the indicated MOI. The cells were harvested 12 h after infection, fractionated, and assessed for indicated
proteins by western blotting (A) and for cytosolic DNA by qPCR (B and C). Data in (B) and (C) are representative of two independent experiments done in triplicate
and are shown relative to control and normalized to Gapdh.
(D and E) RAW264.7 cells were pre-treated with 200 mM 2CMC for 30 min and then infected with MNV1 at an MOI of 5. The cells were harvested 12 h after infection
and assessed for cytosolic DNA by qPCR. Data are representative of two independent experiments done in triplicates and are shown relative to control and
normalized to Gapdh.
(F) RAW264.7 cells were treated with 1 mg/mL EtBr for 48 h, and then assessed for mtDNA by RT-qPCR using the indicated primer sets. Data are representative of
two independent experiments done in triplicate and are shown relative to genomic DNA (GAPDH).
(G–I) EtBr-treated cells in (F) were infected with MNV1 at an MOI of 5. The cells were harvested 9 h after infection and assessed by RT-qPCR. Data are
representative of two independent experiments done in triplicate and are shown relative to control and normalized to Gapdh.
(J) Schematic representation of the experiments carried out in (K).
(K) RAW264.7 cells were infected with MNV1 at the indicated MOIs. The cells were harvested and fractionated 12 h after infection, and DNA was extracted from
the cytosolic and whole cell fractions. Pre-seeded wild-type or STING-KO (KO-1 and KO-2) cells were transfected with normalized amounts of the cytosolic DNA,
harvested 9 h after transfection, and analyzed by qPCR. Data are presented relative to control and normalized to Gapdh. All error bars represent standard error of
the mean. See also Figure S5.

similar to previous reports.43 There was a significant decrease in
the induction of IFN and ISGs in EtBr-treated cells compared
with untreated cells (Figures 5G–5I), suggesting that leaked cyto-
solic DNA contribute to IFN responses in infected cells. To further
confirm the ability of this aberrant cytosolic DNA to induce IFNs, we
transfected normalized amounts of extracted cytosolic DNA into
RAW264.7 cells, and assessed for IFN-b and ISG (viperin) expres-
sion using qPCR (Figure 5J). As shown in Figure S5F, there was a
significant increase in IFN-b and viperin in the cells transfected with
cytosolic DNA from MNV-infected cells, compared with that from
mock-infected cells, in a dose-dependent manner. This increased
induction of IFN-b was seen to be STING dependent, as the induc-
tion of IFN-b in STING
/
cells transfected with cytosolic DNA was
greatly attenuated (Figure 5K). Altogether, our data indicate that

Cell Reports 42, 112179, March 28, 2023 7

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the leakage of genomic and mtDNA into the cytosol of MNV-in-
fected cells activates the induction of IFNs.
The norovirus NS4 protein promotes the accumulation
of cytosolic DNA
Leakage of DNA into the cytosol of cells occurs in disparate ways.
For example, mtDNA leakage in A549 cells was shown to occur
downstream of IL-1b signaling.18 Treatment of these cells with
IL-1b was sufficient to cause mtDNA leakage into the cytosol,
activation of IRF3, and a resultant induction of IFNs and ISGs. Since
MNV infection has recently been shown to induce IL-1b secre-
tion,44 we hypothesized that this could, therefore, potentially
explain the leakage of mtDNA in MNV-infected cells. To determine
if this is the case, RAW264.7 cells were pre-treated with an IL-1
receptor antagonist (IL-1RA), IL-1b, or both, and subsequently
infected with MNV1 at an MOI of 5. The cells were harvested 12 h
after infection and analyzed for IFN-b and viperin expression using
8 Cell Reports 42, 112179, March 28, 2023
Article
Figure 6. NS4 proteins of noroviruses pro-
mote accumulation of cytosolic DNA
(A and B) HEK293T-CD300lf cells were infected
with MNV1 at an MOI of 5. The cells were
harvested 10 h after infection and assessed for
cytosolic DNA by qPCR. Data are presented rela-
tive to mock-infected cells and normalized to their
corresponding whole cell fractions.
(C) Schematic representation of the MNV genome
indicating the different proteins encoded by the
four open reading frames.
(D–G) HEK293T-CD300lf cells were transfected
with Flag-tagged construct plasmids of the
indicated proteins. The cells were harvested 24 h
after transfection and assessed for protein
expression by western blotting, and cytosolic DNA
by qPCR. All qPCR data are presented relative to
GFP and normalized to their corresponding whole
cell fractions. All error bars represent standard
error of the mean. See also Figures S6 and S7.
qPCR. As shown in Figures S6A and S6B,
we neither observed any significant
decrease in IFN-b or viperin induction in
the presence of IL-1RA, nor did we see
any induction of IFN-b or viperin in cells
treated with IL-1b. Additionally, while
treatment of A549 cells with IL-1b did
induce expression of IL-6, with a decre-
ase in expression seen in cells pre-treated
with IL-1RA, as expected (Figure S6C),
in our hands there was no increase in IFN-
b induction in cells treated with IL-1b
(Figure S6D).
Next, we considered whether any of the
structural and non-structural viral proteins
is the cause of leakage of DNA into the
cytosol. One likely candidate is the
NS1-2 protein that was recently shown
to potentially have a viroporin activity.45
We hypothesized that the NS1-2 protein,
as a viroporin, may disrupt intracellular
calcium homeostasis and eventually lead to mitochondrial
leakage, similar to the M2 protein of influenza A virus and the
2B protein of EMCV.14 Another candidate is the NS5 protein
(VPg), which was shown to be a potential interactor of PARP1
and other DNA repair enzymes in a proteomics assay.46 Seques-
tration of DNA repair enzymes by VPg in the cytosol might lead to
a depletion of the nuclear components of these enzymes, which
could then promote the leakage of genomic DNA into the cytosol.
Indeed, expression of the norovirus VPg in cells leads to cell-cy-
cle arrest,47,48 a phenomenon that is also seen after depletion of
DNA repair enzymes. To determine if any of these is relevant to
norovirus biology, we transfected plasmids encoding the nine in-
dividual proteins of MNV1 into HEK293T-CD300lf cells. We opted
to use these cells because of their high transfection efficiencies
and, like RAW264.7 cells, they show a significant accumulation
of genomic and mtDNA in the cytosol after infection with MNV
(Figures 6A and 6B). Transfected cells were harvested after

Article
24 h and cytosolic DNA was assessed by qPCR. The mouse
norovirus encodes six non-structural proteins (NS1/2-NS7), two
structural proteins (VP1 and VP2), and an accessory protein ex-
pressed from an over-lapping open reading frame (VF1)
(Figure 6C).1 After transfection, we observed a significant in-
crease in cytosolic DNA in cells expressing NS4 compared with
those expressing GFP (Figures 6D and 6E). No increase in
cytosolic DNA was seen in cells expressing the NS1-2 protein,
VPg, or any of the other MNV proteins (Figures 6E, S7A, and
S7B), suggesting that the NS4 protein of MNV mediates accumu-
lation of DNA in the cytosol of MNV-infected cells.
To determine if this function is conserved across the NS4
proteins of noroviruses, we explored leakage of DNA in cells ex-
pressing HuNoV NS4. For this, HEK293T-CD300lf cells were
transfected with plasmids encoding GFP, MNV1 NS4, or HuNoV
GII.4 NS4. The cells were harvested 24 h after transfection, and
cytosolic DNA was assessed by qPCR. While no increase in
cytosolic DNA was detected in GFP-expressing cells compared
with mock, as expected, a significant increase was seen in cells
expressing HuNoV NS4, similar to those expressing MNV1 NS4
(Figures 6F and 6G). Interestingly, we observed a greater propor-
tion of mtDNA in cells expressing HuNoV NS4 compared with
those expressing MNV NS4, although levels of genomic DNA re-
mained largely the same. Altogether, these data indicate that
NS4 proteins of noroviruses are necessary and sufficient for
mediating leakage of genomic and mtDNA into the cytosol.
DISCUSSION
Data suggesting a role for STING in restricting RNA viruses are as
old as the discovery of STING itself, and the first viral proteins
shown to antagonize STING function are in fact encoded by
RNA viruses.17 In this study, we explored the potential role of
STING in the IFN responses to norovirus infection. There was a
substantial impairment of IFN responses and a corresponding in-
crease in viral replication in norovirus-infected primary cells and
cell lines after treatment with small molecule inhibitors of STING,
as well as a decrease in the induction of IFNs and ISGs in noro-
virus-infected STING
/
cells, and in cGAS
/
and IFI16
/

cells. Both cGAS and IFI16 can sense the presence of DNA in
the cytosol and activate an IFN induction signaling cascade up-
stream of STING.5 IFI16 has also been shown to be able to sense
viral RNA,49 positively regulates cGAS-STING signaling,50,51 and
promotes both DNA or RNA virus-induced IFN transcription in
the nucleus.52 Although STING mainly functions as an adapter
protein in the intracellular detection of foreign or mislocalized
DNA, it has been shown to play important roles in the restriction
of some RNA viruses through various independent mechanisms
(reviewed by Maringer et al.,17 Aguirre and Fernandez-Sesma,53
Zevini et al.,54 and Ni et al.55). For example, it has been shown
that STING can promote fusion-mediated IFN induction in cells
infected with influenza A virus in a manner independent of
cGAS,56 and facilitate IFN induction downstream of cGAS in a
manner dependent on the viral M2-mediated leakage of
mtDNA.14 In cells infected with DENV, membrane recruitment
to replication complexes likely leads to leakage of mtDNA that
triggers IFN induction via STING, in a process contingent on
cGAS activation.13 Recently, it was also demonstrated that
ll
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STING can inhibit host and viral translation in cells infected
with a wide variety of RNA viruses in a RIG-I-dependent
manner.16 In addition, at least for Japanese encephalitis virus,
IFN induction largely depends on a RIG-I/STING-dependent
pathway.57 And lastly, as we were preparing this manuscript
for submission, another group also published data that corrobo-
rated ours, showing a role for cGAS and STING in IFN responses
against MNV, using KO and overexpression assays.58
The discovery of the role for the cGAS-STING pathway in the
restriction of norovirus replication is significant, and potentially
broadens the current MDA5/MAVS-centric understanding of
the innate immune restriction of noroviruses to one that also in-
cludes a cGAS/IFI16/STING component. Given that knocking
out single proteins in either pathway profoundly affect the IFN
response (Figure 4), it is possible that the two pathways
contribute to the overall IFN response in a synergistic manner
(Figure 7). Our findings are also useful in explaining previous re-
sults relating to norovirus restriction by IFNs. For instance,
STING has been shown to inhibit the replication of RNA viruses
via translation shutoff.16 Since STING is itself an ISG,59 this could
explain a previously described ability of type I IFNs to inhibit
translation of MNV proteins independent of protein kinase R.60
STING is also involved in inflammasome activation after the
detection of pathogens, and this knowledge could, therefore,
facilitate future studies of the complex relationship between nor-
oviruses and commensal bacteria.61,62 Importantly, this also
potentially explains the discrepancy between in vivo and in vitro
results from studies on IFN responses to the HuNoV.12 For
example, studies in Huh7 and 293FT cells have shown no IFN re-
sponses to HuNoV,63,64 while human challenge studies,65 studies
in animal models,66,67 and in vitro studies in organoids37,68,69 and
replicon-containing cell lines36 have all demonstrated induction
of IFNs after infection. Given that both Huh7 and 293FT cells
have impaired cGAS-STING pathways,32,70,71 our data demon-
strating a role for this pathway in the restriction of noroviruses
harmonizes these various otherwise conflicting data.
Our finding that infection promotes leakage of genomic DNA
is surprising, but not unexpected, given the substantial and
widespread membrane reorganization that occurs in infected
cells during the formation of virus replication complexes.72,73
We have shown that expression of the viral NS4 protein is suf-
ficient to cause this accumulation of DNA in the cytosol. Inter-
estingly, a previous study demonstrated that the NS4 protein is
uniquely able to form membranous complexes when overex-
pressed in cells.73 Whether the ability of NS4 to promote
accumulation of cytosolic DNA is related to its membrane
recruitment function remains to be tested. Indeed, the pres-
ence of nuclear envelope markers on norovirus replication
complexes has not been previously explored. While leakage
of genomic DNA into the cytosol of cells infected with RNA vi-
ruses has not been demonstrated until now, it is seen in cancer
cells19,20 and after irradiation21 or exposure to chemothera-
peutic agents such as etoposide.22 Indeed, the efficacy of
some anticancer drugs has been shown to depend on their
ability to activate STING in this manner.23–25,74 Given the wide-
spread reorganization of host cell architecture during viral repli-
cation, we expect that many more RNA viruses likely trigger
leakage of genomic DNA into the cytosol.
Cell Reports 42, 112179, March 28, 2023 9
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Several RNA viruses encode proteins that counteract STING-
dependent host antiviral responses.17 The Dengue virus NS2B
and Chikungunya virus capsid proteins promote degradation of
cGAS in infected cells, for example.13,15 The influenza A virus
NS1 protein binds to mtDNA and renders it less immunostimu-
latory.14 Further work is required to determine if noroviruses
have mechanisms to counteract or avoid this pathway. One po-
tential target could be the GTPase-activating protein SH3
domain-binding protein 1 (G3BP1). Our group and others
have recently shown that G3BP1 is sequestered within replica-
tion complexes in infected cells.46,75,76 Since it was also
recently shown to contribute to DNA sensing by cGAS,77–79 it
is entirely possible that its sequestration in replication com-
plexes also dampens its contribution to cGAS activation.
Further work is, however, required to determine if this is
the case.
In this work, we have shown a role for cGAS, IFI16, and
STING in the restriction of noroviruses and demonstrated the
role of the host genomic DNA as a DAMP in cells infected
with an RNA virus. We have demonstrated an accumulation
of nuclear DNA and, to a lesser extent, mtDNA in the cytosol
of infected cells, likely driven by the viral NS4 protein. Further
work is required to determine the exact mechanism of this
DNA leakage, as well as potential mechanisms of evasion by
the viruses.
Limitations of the study
One limitation of this study is that while we have shown that the
accumulation of DNA in the cytosol of infected cells contributes
10 Cell Reports 42, 112179, March 28, 2023
Article
Figure 7. Contribution of leaked genomic
and mitochondrial DNA to the host response
to noroviruses in a STING-dependent
manner
The MDA5/NLRP6/MAVS pathway is thought to
play a central role in the detection of PAMPs in
norovirus-infected cells, initiating a signaling
cascade that leads to induction of type I and type III
IFNs. Our data show that accumulation of genomic
and mtDNA in the cytosol, driven by the viral NS4,
likely activates the cGAS/IFI16/STING pathway.
The combined activation of the two pathways is
required for a robust IFN response and restriction
of norovirus replication.
to IFN responses, the respective contri-
butions of genomic and mtDNA to the in-
duction of IFNs remains unclear, and it is
possible that one is more important than
the other in driving the response. Treat-
ment of cells with EtBr, a common
method of depleting mtDNA, leads to a
massive decrease in IFN induction in in-
fected cells, suggesting that mtDNA is
the major inducer in the cytosol. Howev-
er, EtBr is also able to bind other extra-
chromosomal DNA, including leaked
genomic DNA. This, and the sheer
amount of genomic DNA in the cytosol
compared with mtDNA, makes any
conclusion regarding the relative contributions of the two
DNA moieties potentially premature. Further experiments,
such as those using mitochondria-targeted endonucleases,
would be needed to determine the specific roles of each in
this context.
Additionally, although we have shown that the NS4 protein
likely drives accumulation of cytosolic DNA, it is clear that other
factors contribute to this, given that we observed between 10
and 100 times more DNA in the cytosols of cells following infec-
tion compared with exogenous expression of NS4 alone. This
follows a similar pattern to the ability of NS4 to promote the gen-
eration of membranous vesicles when expressed alone,
compared with that seen in infected cells,73 suggesting that
the two functions share the same mechanism. Further advances
in our understanding of the mechanism of membrane recruit-
ment by NS4 will enable the determination of whether the two
processes are related and may help in identifying other factors
involved.
Lastly, while we have observed phosphorylation of STING in
MNV-infected cells by microscopy, we were unable to show
this by western blotting, despite trying several anti-phospho-
STING antibodies. Given that only approximately 20%–30%
of the cells show phosphorylation of STING on microscopy af-
ter infection (Figures 1E and 2J), we think it is likely that the
western blotting is not sensitive enough to detect phosphor-
ylation of STING after infection with MNV, compared with
synthetic DNA transfection for example, where we get consid-
erably higher transfection efficiency and involves a lot more
stimulating DNA.
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Article OPEN ACCESS

STAR+METHODS the results. A.S.J. wrote the initial draft of the manuscript. All authors
were involved in the interpretation of the results and writing of the
manuscript.
Detailed methods are provided in the online version of this paper
and include the following:
DECLARATION OF INTERESTS
d KEY RESOURCES TABLE
I.G.G. currently works for Exscientia. However, this manuscript was submitted
d RESOURCE AVAILABILITY
before their affiliation with the company and Exscientia had no involvement or
B Lead contact
input in the research presented in this paper.
B Materials availability
B Data and code availability Received: November 8, 2021
d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DE- Revised: October 11, 2022
TAILS Accepted: February 12, 2023
B Cell lines Published: March 20, 2023

B Primary cells
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Mouse monoclonal anti-GAPDH Ambion Cat#AM4300; RRID: AB_437392
Rabbit polyclonal anti-GAPDH Protein Tech Cat#10494-1-AP; RRID: AB_2263076
Rabbit polyclonal anti-IRF3 Abclonal Cat#A11373; RRID: AB_2758531
Rabbit polyclonal anti-phospho-IRF3 (S396) Abcam Cat#ab138449
Rabbit monoclonal anti-STAT1 Abcam Cat#ab92506; RRID: AB_2197980
Rabbit monoclonal anti-STING (D2P2F) Cell signaling Cat#13647; RRID: AB_2732796
Rabbit polyclonal anti-NS3 Non-commercial N/A
Rabbit polyclonal anti-VPg Non-commercial N/A
Rabbit polyclonal anti-NS7 Non-commercial N/A
Rabbit polyclonal anti-MED1 Bethyl Labs Cat#A300-793A; RRID: AB_577241
Rabbit polyclonal anti-TOM70 (H-117) Santa Cruz Cat#sc-366282
Rabbit monoclonal anti-phospho- Cell signaling Cat#50907S; RRID: AB_2827656
STING (S366) (E9A9K)
Mouse monoclonal anti-dsRNA (J2) Cell signaling Cat#76651
Alexa-flour 488 goat polyclonal Invitrogen Cat#A-11029; RRID: AB_2534088
anti-mouse IgG (H + L)
Alexa-flour 568 goat polyclonal Invitrogen Cat#A-11011; RRID: AB_143157
anti-rabbit IgG (H + L)
Bacterial and virus strains
MNV1 (MNV-1.CW1) Chaudhry et al.80 GenBank accession #: DQ285629.1
26
MNV1 M1 McFadden et al. N/A
Biological samples
Mouse: Bone marrow cells (C57BL/6 mice) N/A N/A
Human: J2 biopsy-derived organoids Ettayebi et al.37 N/A
Human: TI365 biopsy-derived organoids Hosmillo et al.38 N/A
Human: D196 biopsy-derived organoids Hosmillo et al.38 N/A
Chemicals, peptides, and recombinant proteins
Ruxolitinib Invivogen Cat#tlrl-rux
Puromycin Invivogen Cat#ant-pr-1
Hygromycin B Invitrogen Cat#10687010
Poly (I:C) Sigma Cat#P1530
Poly (dA:dT) Sigma Cat#P1537
IL-1RA Peprotech Cat#AF-200-01R
Mouse IL-1b Peprotech Cat#AF-211-11B
Human IL-1b Peprotech Cat#AF-200-01B
DMSO Sigma Cat#D8418-50ML
C-176 Focus Biomolecules Cat#10-4130-0010
(via Tebu-bio UK)
H-151 Focus Biomolecules Cat#10-4132-0010
(via Tebu-bio UK)
20 -C-Methylcytidine (2CMC) Sigma Cat#M4949
RNase Cocktail Enzyme Mix Invitrogen Cat#AM2286
Ethidium Bromide (EtBr) Sigma Cat#E1510
Quanti-luc Gold reagent Invivogen Cat#rep-qlcg1
qPCR Core kit for SYBR Green I, No ROX Eurogentec Cat#RT-SN10-05NR
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
ROX Passive Reference Eurogentec Cat#RT-PARE-03
M-MLV Reverse Transcriptase kit Promega Cat#M1701
Matrigel BD Cat#356231
Advanced DMEM/F12 ThermoFisher Scientific Cat#12634028
Glutamax ThermoFisher Scientific Cat#35050061
Penicillin/Streptomycin ThermoFisher Scientific Cat#15140122
HEPES buffer ThermoFisher Scientific Cat#15630080
WNT conditioned media Non-commercial N/A
R-Spondin conditioned media Non-commercial N/A
Recombinant M Noggin Peprotech Cat#250-38
B27 ThermoFisher Scientific Cat#12587010
n-Acetyl Cysteine Sigma Cat#A9165-5G
Nicotinamide Sigma Cat#N0636-100G
Recombinant mEGF Invitrogen Cat#PMG8043
A8301 Tocris Cat#2939
SB202190 Sigma Cat#S7067-25MG
Primocin Invivogen Cat#ant-pm-1
Y27632 Stem Cell Research Cat#72302
Critical commercial assays
PierceTM BCA Protein Assay Kit ThermoFisher Scientific Cat#23227
GenElute Mammalian Total RNA Miniprep Kit Sigma Cat#RTN70
RNeasy Plus Mini Kit Qiagen Cat#74134
QIAquick Nucleotide Removal kit Qiagen Cat#28304
QIAamp DNA Mini Kit Qiagen Cat#51304
CellTiter-Blue cell viability assay Promega Cat#G8081
Deposited data
RNA-seq This paper Sequence reads were deposited
in the NCBI SRA database under
the BioProject accession number
PRJNA771274
Uncropped western blots This paper Publicly available via Mendeley Data,
https://doi.org/10.17632/j6dtbc96xx.1
Experimental models: Cell lines
Human: HEK293T Susanna M. Colaco RRID: CVCL_0063
(University of Cambridge, UK)
Human: HeLa Susanna M. Colaco RRID: CVCL_0030
(University of Cambridge, UK)
Human: HEK293T-CD300lf This paper N/A
Human: HeLa-CD300lf This paper N/A
Human: HGT Arthur et al.36 N/A
Human: HGT-NV Arthur et al.36 N/A
Hamster: BSRT7 Karl-Klaus Conzelmann N/A
(Ludwid Maximillians
University, Munich, Germany)
Mouse: BV2 Jennifer Pocock RRID: CVCL_0182
(University College London, UK)
Mouse: RAW264.7 Herbert Virgin RRID: CVCL_0493
(Washington University,
St. Louis, MO)
Mouse: RAW264.7 STING-KO-1 This paper N/A
Mouse: RAW264.7 STING-KO-2 This paper N/A
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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Mouse: RAW-lucia ISG Invivogen Cat#rawl-isg; RRID: CVCL_X596
Mouse: RAW-lucia ISG-KO-MAVS Invivogen Cat#rawl-komavs; RRID: CVCL_A7ZE
Mouse: RAW-lucia ISG-KO-STING Invivogen Cat#rawl-kostg; RRID: CVCL_X597
Mouse: RAW-lucia ISG-KO-MDA5 Invivogen Cat#rawl-komda5; RRID: CVCL_A7ZF
Mouse: RAW-lucia ISG-KO-cGAS Invivogen Cat#rawl-kocgas; RRID: CVCL_5I67
Mouse: RAW-lucia ISG-KO-IFI16 Invivogen Cat#rawl-koif16; RRID: CVCL_5I68
Oligonucleotides
See Table S3 for RT-qPCR Primers used This paper N/A
See Table S4 for genomic and This paper N/A
mitochondrial DNA qPCR Primers used
Recombinant DNA
pT7:MNV-1_30 Rz Chaudhry et al.80 N/A
0
pT7:MNV-1_3 Rz_M1 McFadden et al.26 N/A
pFS669IG (CD300lf) This paper N/A
pMDLg/pRRE Addgene Cat#12251
pRSV-Rev Addgene Cat#12253
pMD2G Addgene Cat#12259
shEGFP Sigma Cat#SHC005
shSTING-3 Sigma Cat#SHCLNG-NM_028261
(TRCN0000346266)
pAJ093IG (FLAG-EGFP) This paper N/A
pFS610IG (FLAG-NS1-2) This paper N/A
pFS611IG (FLAG-NS3) This paper N/A
pFS612IG (FLAG-NS4) This paper N/A
pFS613IG (FLAG-NS5) This paper N/A
pFS614IG (FLAG-NS6) This paper N/A
pFS615IG (FLAG-NS7) This paper N/A
pFS616IG (FLAG-VF1) This paper N/A
pFS621IG (FLAG-VP1) This paper N/A
pFS617IG (FLAG-VP2) This paper N/A
pAJ124IG (FLAG-GII.4-NS4) This paper N/A
pAJ221IG (mouse STING sgRNA) This paper N/A
pAJ222IG (FLAG-tagged This paper N/A
CRISPR-resistant mouse STING)
Software and algorithms
Prism 9.5.0 Graph Pad https://www.graphpad.com/
scientific-software/prism/;
RRID: SCR_002798
ImageJ FijiSc https://fiji.sc/; RRID: SCR_002285
Image Studio Lite 5.2 Li-COR https://www.licor.com/bio/
image-studio-lite/; RRID: SCR_013715
Clustal Omega EMBL-EBI https://www.ebi.ac.uk/Tools/
msa/clustalo/; RRID: SCR_001591
Snapgene 4.2 Dotmatics https://www.snapgene.com/;
RRID: SCR_015052

RESOURCE AVAILABILITY

Lead contact
Any request for information, reagents, and other resources used in this paper should be directed to Ian G. Goodfellow (ig299@cam.
ac.uk).

Cell Reports 42, 112179, March 28, 2023 17

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Materials availability
Cell lines and plasmids generated in this paper are available upon request from the lead contact after completion of a Materials Trans-
fer Agreement (MTA).

Data and code availability

d RNA-seq data generated in this paper have been deposited in the NCBI Sequence Read Archive Database (SRA; https://www.
ncbi.nlm.nih.gov/sra) under the BioProject accession number PRJNA771274 (GenBank SRA: PRJNA771274). Uncropped
western blots are publicly available through Mendeley Data: https://doi.org/10.17632/j6dtbc96xx.1.
d This paper does not report original code.
d Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Cell lines
RAW264.7, BV2, HEK293T, HeLa, HGT, and HGT-NV cells were maintained at 37 C in complete Dulbecco’s Modified Eagle Medium
(DMEM, Sigma Aldrich) containing 4500 mg/ml glucose, sodium bicarbonate, and sodium pyruvate, and supplemented with 10%
heat-inactivated Fetal Bovine Serum (HyClone), 10U/ml of penicillin, 100 mg/mL of streptomycin, 2mM L-glutamine (Sigma Aldrich),
and non-essential amino acids (Sigma Aldrich). Media used for HGT-NV cells was also supplemented with 500mg of G418. HEK293T
and HeLa cells used in this work were generously provided by Dr Susanna M. Colaco (University of Cambridge). BSR-T7 cells, a kind
gift from Karl-Klaus Conzelmann (Ludwid Maximillians University, Munich) derived from Baby Hamster Kidney (BHK) cells and
expressing the T7 RNA polymerase, were maintained in complete DMEM supplemented with 0.5 mg/mL G418 (Invivogen).
RAW-Lucia ISG wild type (Invivogen, rawl-isg), MAVS-KO (Invivogen, rawl-komavs), STING-KO (Invivogen, rawl-kostg), MDA5-KO
(Invivogen, rawl-komda5), cGAS-KO (Invivogen, rawl-kocgas), and IFI16-KO (Invivogen, rawl-koif16) were purchased from Invivogen.

Primary cells
Bone marrow-derived macrophages (BMDMs) were differentiated from bone marrow cells of C57BL/6 mice as previously
described.81 Briefly, bone marrow cells were seeded on non-treated culture plates in complete DMEM supplemented with 10%
CMG14 culture supernatant which contains M-CSF. Fresh medium was added every 3 days and cells were harvested and used
for experiments on day 9 or 10. This work was carried out in accordance with regulations of The Animals (Scientific Procedures)
Act 198682 and the ARRIVE guidelines.83 All procedures were approved by the University of Cambridge Animal Welfare and Ethical
Review Body (AWERB) and the UK Home Office and carried out under the Home Office project licence PPL 70/7689.

Human intestinal organoids

Established human intestinal organoids TI365 (terminal ileum) and D196 (duodenal) received from Professor Matthias Zilbauer
(Department of Pediatrics, University of Cambridge) were generated from normal small intestinal biopsy samples collected from pa-
tients during routine endoscopy at Cambridge University Hospitals NHS Foundation Trust (Cambridge, UK) following local ethics
committee approval (REC-12/EE/0482) and informed consent.37,84 The J2 (jejunal) human intestinal organoids culture was a kind
gift from Professor Mary K. Estes (Baylor College of Medicine, Houston, Texas, USA), and has been described previously.38 All or-
ganoid samples received were anonymized, and hence age and sex information was not provided.
Culture conditions for the biopsy-derived human intestinal organoids have been previously described.37 Briefly, the organoids were
grown within matrigel basement membrane matrix domes (BD, 356,231) as undifferentiated 3D cultures in proliferation media made up
of Advanced DMEM/F12 (ThermoFisher Scientific, 12,634,028) supplemented with 2mM Glutamax (ThermoFisher Scientific,
35,050,061), 100 units/mL Penicillin/Streptomycin (ThermoFisher Scientific, 15,140,122), 10 mM HEPES buffer (ThermoFisher Scientific,
15,630,080), 2x WNT conditioned media (non-commercial), 5x R-Spondin conditioned media (non-commercial), 100 ng/mL recombi-
nant M Noggin (Peprotech, 250-38), 1x B27 (ThermoFisher Scientific, 12,587,010), 1.25 mM n-Acetyl Cysteine (Sigma, A9165-5G),
10 mM Nicotinamide (Sigma, N0636-100G), 50 ng/mL recombinant mEGF (Invitrogen, PMG8043), 2 mM A8301 TGF-beta inhibitor (Toc-
ris, 2939), 3 mM SB202190 p38 inhibitor (Sigma, S7067-25MG), 100 mg/mL Primocin (Invivogen, ant-pm-1), and 10 mM Y27632 ROCK
inhibitor (Stem Cell Research, 72,302). Media was changed 3 times a week, and the organoids were split once every 1 to 2 weeks.

METHOD DETAILS

Plasmids
Plasmids used in this work are listed in Table S1.

Lentivirus transduction
For shRNA transduction, Mission shRNA plasmids (Sigma Aldrich) were transfected together with pMDLg/pRRE, pRSV-Rev, and
pMD2.G plasmids into HEK293T cells using Lipofectamine 2000. Pooled lentiviral supernatants harvested on days 2 and 3 were

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used to infect RAW264.7 cells. Puromycin (Invitrogen) selection was started 72 h post-infection. The cells were cultured in 2 mg/mL
puromycin until all the control cells were dead and were then maintained in 5 mg/mL puromycin.
For CD300lf lentiviral transduction, the pFS669IG plasmid encoding the mouse CD300lf was transfected together with pMDLg/
pRRE, pRSV-Rev, and pMD2.G plasmids into HEK293T cells using Lipofectamine 2000. Pooled lentiviral supernatants harvested
on days 2 and 3 were used to infect HeLa and HEK293T cells. CD300lf-transduced HeLa and HEK293T cells were subsequently
selected using 100 mg/mL Hygromycin (Invitrogen), starting 72 h post-infection.
For generation of STING
/
RAW264.7 cells, the LentiCRISPRv2-based plasmid pAJ221IG, co-encoding the Cas9 nuclease and
single guide RNA targeting mouse STING (sequence: AGCAAAACATCGACCGTGC, from Storek et al.35), was transfected together
with pMDLg/pRRE, pRSV-Rev, and pMD2.G plasmids into HEK293T cells using Lipofectamine 2000. Pooled lentiviral supernatants
harvested on days 2 and 3 were used to infect RAW264.7 cells. Puromycin (Invitrogen) selection was started 72 h post-infection. The
cells were cultured in 2 mg/mL puromycin until all the control cells were dead and were then maintained in 5 mg/mL puromycin. Single
cells were FACS-sorted into individual wells of 96-well plates containing conditioned media by the NIHR Cambridge BRC Cell Phe-
notyping Hub. Clones of cells were screened 6 weeks later by western blotting. Clones 14 (KO-1) and 22 (KO-2) were used in the
current study. As indicated in Table S6, the KO-1 cells were found to express three mutated alleles of the STING gene on RNAseq,
with the first allele bearing a deletion of 239 nucleotides leading to a premature termination codon, and the second and third alleles
having in-frame deletions of 243 and 348 nucleotides, respectively. Transcripts from a single allele were detected in the KO-2 cells
with one nucleotide deletion leading to a premature termination codon. No reads corresponding to the wild-type STING allele were
detected in either STING-KO clone.
For STING complementation in knockout cells, the pAJ222IG plasmid encoding an N-terminal flag-tagged CRISPR/Cas9-resistant
murine STING was generated by introducing the following silent mutations at the CRISPR/Cas9 target site using overlap extension
PCR: G762A, A765G, C768T, C771T, C774T, C775A, and T777G (see Figure S3B). cDNA generated from RNA extracted from
RAW264.7 cells was used as template. Lentiviruses made from HEK293T cells were used to transduce STING knockout
RAW264.7 clones 14 (KO-1) and 22 (KO-2) cells, using the same protocol as described above. Selection using 100 mg/mL hygromycin
was started 72 h post-transduction.

Reverse genetics
The MNV1 virus was prepared via reverse genetics as previously described.80,85 Briefly, 1.5x106 BSR-T7 cells were seeded in a
6-well plate and incubated at 37 C for 3 h. The cells were then infected with Fowlpox virus (FPV)-T7 at an MOI of 0.5 pfu/cell and
incubated at 37 C for 2 h. Then, 1mg of the pT7:MNV-1_30 Rz or the pT7:MNV-1_30 Rz_M1 MNV cDNA clones (for the wild type or
VF1-deficient M1 mutant, respectively) were transfected using Lipofectamine 2000, according to the manufacturer’s instructions.
The plate was incubated at 37 C for 2 days, freeze-thawed once (at
80 C overnight or longer), and titred by TCID50.

TCID50
TCID50 by cytopathic effect (CPE) was carried out as previously described.80 Briefly, 1:10 serial dilutions of the virus preparations
were made in cell culture media and aliquoted into wells of a 96-well plate, each in 4 replicates of 50mL. Then, 2x104 BV2 cells in
100mL of cell culture media was added to each well and the plate was incubated at 37 C for 5 days. The cells were subsequently
assessed for CPE, and TCID50/ml was calculated using the Spearman & Kärber algorithm.86

Cell viability assessment

Cell viability was determined using the CellTiter-Blue assay (Promega), according to the manufacturer’s protocol. Briefly, 20mL of the
CellTiter-Blue reagent was added to cells growing in 100mL media per well of a 96-well plate 4 h before harvest. At the time of harvest,
the fluorescence at 560nm/590nm was read on a SpectraMax i3 plate reader (Molecular Devices).

Cell stimulation and virus infection

Poly(I:C) (P1530, Sigma) and poly(dA:dT) (P1537, Sigma) transfections were carried out on cells pre-seeded overnight in 24-well
plates using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. Animal-free recombinant IL-1RA (AF-200-
01RA), mouse IL-1b (AF-211-11B) and human IL-1b (AF-200-01B) were purchased from Peprotech.
For MNV infection, cells were incubated with the virus inoculum at the appropriate MOI on an end-to-end rotor at 37 C for an hour,
then washed twice with fresh media, transferred to appropriate culture plates, and incubated at 37 C.
Differentiation and infection of biopsy-derived human intestinal organoids has been previously described.37 Briefly, the organoids
were trypsinised and monolayers were seeded onto collagen-coated wells in 48-well plates. Proliferation media was replaced with
differentiation media (proliferation media without WNT conditioned media, R-Spondin conditioned media, Nicotinamide, and
SB202190) 1 day post-seeding. On day 5 post-seeding, human norovirus-positive stool filtrates prepared from stool samples
collected from patients at Addenbrookes Hospital, Cambridge, UK, after obtaining informed consent, and containing 1x106 viral
RNA copies were added to the monolayers and incubated for 2 h to allow optimal adsorption. The inoculum was removed, and mono-
layers were then washed and incubated in differentiated media supplemented with 200mM glycochenodeoxycholic acid (GCDCA,
Sigma, G0759) at 37 C. Where indicated, wells were treated with DMSO (Sigma) or 15mM H-151 (Focus Biomolecules). To enhance
virus replication in the organoid-derived culture system, 5mM Ruxolitinib (Invivogen) was also added following virus inoculation, and

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OPEN ACCESS Article

the drugs were maintained up to 2 days until samples were harvested. Total RNA was extracted (GenElute Mammalian Total RNA Kit,
Sigma-Aldrich), from samples collected at day 0 and day 2 post-infection and analyzed by RT-qPCR.

Small molecule inhibition of STING

For experiments involving STING inhibition, cells were pre-treated in DMSO (Sigma), C-176 (Focus Biomolecules), or H-151 (Focus
Biomolecules) for 2 h before infection or transfection with poly(I:C) or poly(dA:dT), and the drugs are supplemented in the media
onwards until the cells were harvested for endpoint assays.

Inhibition of JAK1/JAK2
Unless indicated otherwise, cells were treated with 10 mM Roxolitinib (Invivogen), with a 30-min pre-treatment prior to infection.

Inhibition of norovirus replication

For assays involving small molecule inhibition of replication, cells were treated with 200mM of 20 -C-Methylcytidine (2CMC) (Sigma
Aldrich, M4949), and were harvested at the indicated timepoints. For experiments involving MNV1, cell were pre-treated with
2CMC 30 min before infection.

Luciferase assay
RAW-Lucia ISG cells were infected as described. Clarified supernatants, harvested at 18 h post-infection, were mixed with the
Quanti-luc Gold reagent (Invivogen, rep-qlcg1) in a 1:1 ratio, and analyzed on a Glomax Navigator Microplate Luminometer
(Promega).

Western blotting
Cells were washed in ice-cold PBS twice, resuspended in RIPA buffer (150mM NaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS,
25mM Tris-HCl pH 7.4) supplemented with a protease inhibitor cocktail (and a phosphatase inhibitor cocktail when phospho-proteins
were of interest), and kept on ice for 20 min. The sample was pipetted up and down several times and was centrifuged immediately at
10,000 x g for 10 min at 4 C. The supernatant was transferred to a new tube and the pellet was discarded. The sample was quantified
using the BCA assay (Thermo Scientific) according to the manufacturer’s recommendations. The sample was then mixed with SDS
polyacrylamide gel electrophoresis (PAGE) sample buffer (2% SDS, 10% glycerol, 0.002% bromophenol blue, 0.0625M Tris-Cl pH
6.8, 5% 2-mercaptoethanol), heated at 95 C for 5 min, and kept at
20 C or used immediately for SDS PAGE. Transfers were made
onto 0.45mm nitrocellulose membranes. The membranes were blocked in 5% milk PBST for 1 h at room temperature, and the primary
and secondary antibodies were incubated at 4 C overnight and 1 h at room temperature respectively, with three 5-min washes in
between incubations. The membranes were subsequently scanned on an Odyssey CLx imager (LI-COR) and the results were
analyzed using the Image Studio Lite software version 5.2.5 (LI-COR). Antibodies used in this work are listed in Table S2.

Relative RT-qPCR
RNA extraction with on-column DNAse treatment were done using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich)
according to the manufacturer’s protocol. cDNA was synthesized using the M-MLV Reverse Transcriptase (Promega), according to
the manufacturer’s protocol. qPCR was carried out using a 2X SYBR Green mastermix containing 2.5mM MgCl2, 400mM dNTPs,
1/10,000 SybrGreen (Molecular Probes), 1M Betaine (Sigma), 0.05U/ml of Gold Star polymerase (Eurogentec), 1/5 10X Reaction
buffer (750 mM Tris-HCl pH 8.8, 200 mM [NH4]2SO4, 0.1% [v/v] Tween 20, Without MgCl2), and ROX Passive Reference buffer
(Eurogentec), and ran on a ViiA 7 Real-Time PCR System (ThermoFisher Scientific), with a 15-s 95 C denaturation step and a
1-min 60 C annealing/extension step for 40 cycles. Relative gene expression was calculated using the Livak method (DDCt) relative
to mock-transfected conditions,87 and normalized to a house keeping gene (Gapdh for all the mouse samples, and b-actin for the
human samples). Primers used in this work are listed in Table S3.

RNA sequencing and data analysis

Wild type, STING-KO clone 14 (KO-1), and STING-KO clone 22 (KO-2) RAW264.7 cells were infected with MNV1 at an MOI of 5. Cells
were harvested 9 h post-infection, and total RNA was extracted using Qiagen RNeasy plus kit (Qiagen) which has a genomic DNA
removal step. RNA samples were sent to Novogene UK, where sample quality control, library preparation, and sequencing were car-
ried out. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies) and
sample concentration and purity was determined using a NanoDrop spectrophotometer (Thermofisher). For library preparation,
enrichment of poly(A) (+) mRNA was carried out using poly-T oligo-attached magnetic beads. Next, first and second strand cDNA
synthesis were completed using M-MLV Reverse Transcriptase, RNase H Minus (M-MLV RT [H–]) and DNA polymerase I with RNAse
H, respectively, followed by adaptor ligation and purification with Ampure XP beads (Beckman Coulter). PCR products, generated
using Phusion High-Fidelity DNA polymerase, and Universal PCR primers and Index (X) Primers, were then bead purified and as-
sessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Index-coded samples were clustered on a cBot Cluster Generation
System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) and sequenced using an Illumina Novaseq platform, generating 150 bp
paired-end reads to a depth ranging from 65 to 99 million reads per sample.

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Data analysis was performed as in Hosmillo et al.37 Briefly, raw reads were first inspected with FastQC. Sequencing adapters and
low quality reads were then removed by the use of Trimmomatic version 0.39. Transcript quantification was performed using kallisto
against the mouse transcriptome derived from the GRCm38.p6 genome assembly. Genes with less than five read counts per million
were considered as unexpressed and excluded from downstream analysis. Differential gene expression analysis was obtained using
the edgeR pipeline. Gene ontology enrichment analysis was performed using EnrichR. Sequence reads were deposited in the NCBI
Sequence Read Archive Database (SRA; https://www.ncbi.nlm.nih.gov/sra) under the BioProject accession number PRJNA771274.

Cell fractionation and cytosolic DNA assessment

Cell fractionation for cytosolic DNA assessment was carried out using a protocol modified from Moriyama et al.14 Briefly, the cells
were washed in 1 mL of ice-cold PBS. Cells were then resuspended in 600mL digitonin lysis buffer (150 mM NaCl, 50 mM HEPES
pH 7.4, and 20 mg/mL digitonin), out of which 100mL was set aside for analysis by Western blot, and 100mL was set aside as the whole
cell fraction. The remaining 400mL was centrifuged at 1,000 x g for 3 min, and the supernatant transferred to a new tube. This was
repeated twice, and then the supernatant was centrifuged at 17,000 x g for 10 min and transferred into a new tube. An RNAse diges-
tion was carried out on this cytosolic fraction and the whole cell fraction by adding 2.5 mL each (1.25 units) of the RNase Cocktail
Enzyme Mix (Invitrogen, AM2286) and incubated at 37 C for 2 h. DNA was then extracted from the cytosolic fraction using the
QIAquick Nucleotide Removal kit (QIAGEN) and from the whole cell fraction using the QIAamp DNA Mini Kit (QIAGEN). Both cytosolic
and whole cell fractions were eluted in 100mL and diluted 1:10 in water before assessment by qPCR. Primers used for qPCR are listed
in Table S4.

Mitochondrial DNA depletion

RAW264.7 cells were maintained in media supplemented with 1 mg/ml of Ethidium Bromide (EtBr) (Sigma-Aldrich, E1510) for 48h. For
assessment of mitochondrial DNA depletion, total DNA was extracted using the QIAamp DNA Mini Kit (Qiagen), according to the
manufacturer’s protocol.

Confocal microscopy
Cells on coverslips were fixed by incubation with 4% paraformaldehyde in PBS for 20 min, and then permeabilised by incubation with
0.2% Triton X-100 in PBS for 10 min at room temperature. This was followed by a 1-h incubation in blocking buffer (5% goat serum,
1% BSA in PBST) at room temperature, and an overnight incubation with primary antibodies (diluted in blocking buffer) at 4 C. In-
cubation with secondary antibodies (diluted in blocking buffer) was then carried out for an hour at room temperature, with three
5-min washes in PBST before and after. Coverslips were mounted on slides using MOWIOL containing the DAPI nuclear stain,
and cured overnight at room temperature. Images were acquired using a Leica SP5 confocal microscope. Antibodies used for
confocal microscopy are indicated in Table S2.

QUANTIFICATION AND STATISTICAL ANALYSIS

Prism 9.5 (Graph Pad) was used for all statistical analyses, and one-way repeated measures ANOVA with Bonferroni multiple com-
parisons tests was applied to determine statistical significance, unless where indicated otherwise. In all cases, ‘ns’, *, **, ***, and ****
are used to denote p > 0.05, p % 0.05, p % 0.01, p % 0.001 and p % 0.0001 respectively. The ImageJ software was used for all
confocal micrograph preparation and Image Studio Lite 5.2 was used for Western blot quantification. Clustal Omega was used
for all sequence alignments, and Snapgene 4.2 was used for primer design and cloning strategies. The image on Figure 7 and the
graphical abstract were both created with BioRender.com.

Cell Reports 42, 112179, March 28, 2023 21