core.ac.uk · 2017-03-09 · double-stranded RNA: dsRNA), 5’-ppp dsRNA, and poly AdT (p-AdT; as...

The cyclic GMP-AMP synthetase-STING signaling pathway is required for both

the innate immune response against HBV and the suppression of HBV assembly

Hiromichi Dansako1, Youki Ueda1, Nobuaki Okumura1, Shinya Satoh1, Masaya

Sugiyama2, Masashi Mizokami2, Masanori Ikeda1,3, Nobuyuki Kato1

1 Department of Tumor Virology, Okayama University Graduate School of Medicine,

Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama

700-8558, Japan

2 Research Center for Hepatitis and Immunology, National Center for Global Health

and Medicine, 1-7-1, Kohnodai, Ichikawa 272-8516, Japan

3 Department of Persistent and Oncogenic Viruses, Center for Chronic Viral Disease,

Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1

Sakuragaoka, Kagoshima 890-8544, Japan

Running title: The suppression of HBV assembly through cGAS-STING signaling

pathway

Correspondence

N. Kato, Department of Tumor Virology, Okayama University Graduate School of

Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku,

Okayama 700-8558, Japan.

Fax: +81 86 235 7392

Tel: +81 86 235 7385

E-mail: [email protected]

Abbreviations

cGAS, cyclic GMP-AMP synthetase; HBV, hepatitis B virus; dsDNA, double-stranded

DNA; HCV, hepatitis C virus; rcDNA, relaxed circular DNA; cccDNA, covalently

closed circular DNA; ssRNA, single-stranded RNA; pgRNA; pregenomic RNA; NTCP,

sodium taurocholate cotransporting polypeptide; ssDNA, single-stranded DNA; HSV-1,

herpes simplex virus type 1; VACV, vaccinia virus; HIV, human immunodeficiency

virus; kb, kilobase; ORF, open-reading frame; IFN, interferon; ISG, IFN-stimulated

gene; siRNA, small interfering RNA

Keywords

Antiviral response; hepatitis B virus; innate immune response; cGAS-STING signaling

pathway; viral assembly

Abstract

During viral replication, the innate immune response is induced through the recognition

of viral replication intermediates by host factor(s). One of these host factors, cyclic

GMP-AMP synthetase (cGAS), was recently reported to be involved in the recognition

of viral DNA derived from DNA viruses. However, it is uncertain whether cGAS is

involved in the recognition of hepatitis B virus (HBV), which is a hepatotropic DNA

virus. In the present study, we demonstrated that HBV genome-derived dsDNA induced

the innate immune response through cGAS and its adaptor protein, STING, in human

hepatoma Li23 cells expressing high levels of cGAS. In addition, we demonstrated that

HBV infection induced ISG56 through the cGAS-STING signaling pathway. This

signaling pathway also showed an antiviral response towards HBV through the

suppression of viral assembly. From these results, we conclude that the cGAS-STING

signaling pathway is required for not only the innate immune response against HBV but

also the suppression of HBV assembly. The cGAS-STING signaling pathway may thus

be a novel target for anti-HBV strategies.

Introduction

Hepatitis B virus (HBV) is an enveloped double-stranded DNA (dsDNA) (3.2 kilobase;

kb) virus classified into the Hepadnaviridae family. Chronic hepatitis is caused not only

by hepatitis C virus (HCV) but also by HBV, and then progresses to liver cirrhosis and

hepatocellular carcinoma. Since approximately 350 million people are infected with

HBV worldwide, HBV infection is a serious global health problem [1, 2]. These

diseases are tightly associated with inflammation caused by persistent HBV infection in

the liver. To suppress the progression of hepatic diseases, it is necessary to prevent

persistent HBV infection. However, HBV is known to evade the host innate immune

response for persistent infection [3, 4].

HBV has a relaxed circular DNA (rcDNA) as a viral genome [5, 6]. Following the

invasion of HBV to hepatocytes, intracellular HBV rcDNA is converted to a covalently

closed circular DNA (cccDNA) by DNA repair machinery. The transcription from the

cccDNA is controlled by liver-enriched transcriptional factors [7, 8]. Four viral

single-stranded RNAs (ssRNAs; 3.5, 2.4, 2.1, and 0.7 kb) are transcribed from HBV

cccDNA. Among these viral ssRNAs, 3.5 kb RNA functions as an HBV pregenomic

RNA (pgRNA). By the reverse transcriptase activity of HBV DNA polymerase, a

negative-stranded DNA is synthesized from HBV pgRNA, and subsequently HBV

rcDNA is formed by the synthesis of positive-stranded DNA from negative-stranded

DNA. HBV rcDNA contains four open-reading frames (ORFs) encoding preS1/preS2/S

(S), precore/core (C), polymerase (P), and X antigen genes (known as S gene, C gene, P

gene, and X gene, respectively). S gene encodes three envelop proteins of different

sizes; Large S (prepreS1/preS2/S), Middle S (preS2/S), and Small S (S), respectively. P

gene encodes DNA polymerase responsible for the reverse transcription of HBV

pgRNA [5, 6].

During the life cycle of HBV, viral dsDNA (rcDNA and cccDNA), viral

single-stranded DNA (ssDNA; positive or negative strand) and four viral ssRNAs are

produced as viral replication intermediates. Since HBV replication contains the step of

reverse transcription, it has been predicted that a DNA:RNA duplex is produced as a

viral replication intermediate [5]. Very recently, the RNA sensor RIG-I was reported to

induce the innate immune response through recognition of the 5’-εregion of HBV

pgRNA [9]. However, the host recognition mechanism toward HBV DNA remains

unclear. One of the DNA sensors, cyclic GMP-AMP synthetase (cGAS), was recently

reported to recognize cytosolic DNA, and to induce the activation of transcription factor

IRF-3 and subsequently the production of interferon (IFN)- β and numerous

IFN-stimulated genes (ISGs) such as ISG56 (also known as IFIT1) and ISG15 in a

STING (adaptor protein of cGAS)-dependent manner (this sequence is known as the

cGAS-STING signaling pathway) [10]. The cGAS-STING signaling pathway was also

required for the recognition of infection with several viruses such as herpes simplex

virus type 1 (HSV-1) [11], vaccinia virus (VACV) [11], and human immunodeficiency

virus (HIV) [12]. Therefore, we presumed that the cGAS-STING signaling pathway is

involved in the recognition of HBV infection. To evaluate this presumption, we

examined whether the cGAS-STING signaling pathway is required for the recognition

of HBV using human hepatoma Li23 cells [13] and HBV-replicating HepG2.2.15 cells

[14].

Here, we show that the cGAS-STING signaling pathway is required for the innate

immune response against HBV, and that this signaling pathway is also involved in the

suppression of HBV assembly.

Results

The synthetic analogues of Z-form DNA, p-dGdC, induced ISG56 in Li23 cells

Host cells induce the innate immune response through the recognition of viral

replication intermediates. Very recently, the RNA sensor, RIG-I, was reported to

recognize the 5’- region of HBV pgRNA [9]. However, the host recognition

mechanism toward HBV DNA is uncertain. To clarify this mechanism, hepatic cell lines

showing an innate immune response to HBV DNA were required. Therefore, we first

evaluated the capabilities of synthetic DNA or RNA analogues such as poly dAdT

(p-dAdT; as B-form DNA), poly dGdC (p-dGdC; as Z-form DNA), poly IC (p-IC; as

double-stranded RNA: dsRNA), 5’-ppp dsRNA, and poly AdT (p-AdT; as DNA:RNA

duplex) as indices of ISG56 induction [15]. Having checked several human hepatic cell

lines, we found that only Li23 (human hepatoma cell line) and NKNT-3 (human

immortalized hepatocyte cell line) showed the innate immune responses to both p-dGdC

and p-dAdT (Table 1). These findings also suggest that the mechanism of recognition to

p-dGdC (Z-form DNA) is distinct from that to p-dAdT (B-form DNA), and that the host

factor or factors involved in the recognition of p-dGdC is or are expressed only in Li23

and NKNT-3 cells. Since Li23 cells have been well genetically characterized [13,

16-18], we used the Li23 cell line in the following study.

P-dGdC triggered the cGAS-STING signaling pathway in Li23 cells

We next tried to identify the host factor(s) required for the recognition of p-dGdC in

Li23 cells. HCV nonstructural protein 3 and 4A (NS3/4A) are known to prevent the

RIG-I/IPS-1-mediated signaling pathway through the cleavage of IPS-1 [19, 20].

Therefore, using Li23 cells stably expressing exogenous HCV NS3/4A (designated Li23

NS3/4A cells), we examined whether p-dGdC and p-dAdT induced ISG56 through the

RIG-I/IPS-1-mediated signaling pathway. The results revealed that NS3/4A did not

prevent ISG56 induction by p-dAdT, although that by p-IC was absolutely prevented by

NS3/4A (left and central panels in Fig. 1A). NS3/4A also did not inhibit ISG56

induction by p-dGdC (right panel in Fig. 1A). These results suggest that the

dsDNA-triggered signaling pathway is distinct from the RIG-I/IPS-1-mediated

signaling pathway in Li23 cells.

On the other hand, it was reported that cytosolic DNA was recognized by cGAS and

subsequently induced the innate immune response [10-12]. Therefore, we examined the

expression levels of cGAS in several human hepatic cell lines, including Li23. We

obtained the interesting result that cGAS mRNA was highly expressed in only NKNT-3

and Li23 cells (Fig. 1B). This result is in accord with the former result that only

NKNT-3 and Li23 cells were able to recognize p-dGdC (Table 1). Therefore, we next

examined whether cGAS is required for the p-dGdC-triggered signaling pathway. The

results revealed that p-dGdC-triggered ISG56 induction was remarkably prevented in

cGAS-knockdown Li23 cells (Fig. 1C and right panel in Fig. 1D). Since p-dAdT, but

not p-IC-triggered ISG56 induction was also significantly prevented in the

cGAS-knockdown cells (central and left panels in Fig. 1D), we inferred that the

p-dAdT-triggered signaling pathway was also partially regulated by cGAS in Li23 cells.

It has been also reported that cGAS activates its downstream transcription factor,

IRF-3, and subsequently induces IFN-β and ISGs through the association with its

adaptor protein, STING [10]. Therefore, we examined whether STING and IRF-3 were

required for the p-dGdC- or p-dAdT-triggered signaling pathway in Li23 cells. The

results revealed that STING- (Fig. 2A) and IRF-3-knockdown (Fig. 2B) Li23 cells

greatly weakened both p-dGdC- and p-dAdT-triggered ISG56 induction (Figs. 2C and

2D). Taken together, these results lead us to suggest that the cGAS-STING signaling

pathway is required for the recognition of p-dGdC, and that this pathway is partially

involved in the recognition of p-dAdT.

HBV triggered the cGAS-STING signaling pathway

It has been reported that the cGAS-STING signaling pathway functions as an antiviral

sensor for infection with DNA viruses such as HSV-1 and VACV [11]. Therefore, we

used several synthetic dsDNAs, including HBV-derived dsDNA, to examine whether

the cGAS-STING signaling pathway is required for the recognition of HBV dsDNA

(Fig. 3A). Although ISG56 was reproducibly induced by HBV-derived synthetic dsDNA

as well as p-dGdC and VACV- or HSV-derived dsDNA, these inductions were

remarkably prevented in the cGAS-knockdown (left panel of Fig. 3B) and

STING-knockdown (right panel of Fig. 3B) Li23 cells. In addition, HBV positive- and

negative-stranded ssDNA did not induce ISG56 in Li23 cells (Fig. 3C). These results

suggest that the cGAS-STING signaling pathway is required for the recognition of HBV

dsDNA.

It is important to examine whether the cGAS-STING pathway functions as a sensor

for viral dsDNA occurring in HBV-reproducing cells. HBV-replicating HepG2.2.15

cells are currently used for the study of the life cycle of HBV worldwide [14]. Since

both HepG2.2.15 cells and their parental HepG2 cells exhibit defective cGAS

expression and low STING expression compared with those in Li23 cells (Fig. 4A), we

first prepared HepG2 and HepG2.2.15 cells stably expressing both exogenous cGAS or

cGAS GSAA (the inactive mutant of cGAS), and STING (designated HepG2

cGAS/STING, HepG2 cGAS GSAA)/STING, HepG2.2.15 cGAS/STING, and

HepG2.2.15 cGAS GSAA)/STING cells) (Fig. 4B). The results showed that ISG56 (Fig.

4C) and ISG15 (Fig. 4D) were induced only in HepG2.2.15 cGAS/STING cells.

We next examined whether the cGAS-STING pathway functions as a sensor for

viral dsDNA occurring in HBV-infecting cells. The sodium taurocholate cotransporting

polypeptide (NTCP) was recently shown to act as a functional receptor for HBV [21].

Since HepG2 cells showed defective NTCP expression, we prepared HepG2/NTCP-myc

cells stably expressing both exogenous cGAS or cGAS GSAA, and STING in addition

to NTCP containing myc tag (designated HepG2/NTCP-myc cGAS/STING and

HepG2/NTCP-myc cGAS GSAA/STING cells) for an experiment on HBV infection.

We also prepared an HBV inoculum by concentrating the supernatant of HepG2.2.15

cells (designated HBVcc; cell-cultured HBV). At 13 days after HBVcc infection, we

observed that ISG56 was induced in HepG2/NTCP-myc cGAS/STING cells, but not in

HepG2/NTCP-myc cGAS GSAA/STING cells (Fig. 4E). These results suggest that

HBV triggered the cGAS-STING signaling pathway.

The cGAS-STING signaling pathway showed an antiviral response towards HBV

through the suppression of viral assembly

To evaluate the antiviral response toward HBV through the cGAS-STING pathway, we

first examined the level of HBV RNA in HepG2.2.15 cGAS/STING cells. Northern blot

analysis revealed that the 3.5 kb HBV RNA (corresponding to HBV pgRNA) was

slightly reduced in HepG2.2.15 cGAS/STING cells compared with the level in control

cells (Fig. 5A). Quantitative analysis also revealed that the levels of HBV total

transcript and pgRNA were significantly reduced in HepG2.2.15 cGAS/STING cells,

compared with the control cells. Such a reduction was not observed in HepG2.2.15

cGAS GSAA/STING cells (Fig. 5B). We further examined the levels of HBV RNA after

HBVcc infection. At 13 days after infection, we again observed that the total amount of

HBV transcript was significantly reduced only in HepG2/NTCP-myc cGAS/STING

cells, compared with the control cells or HepG2/NTCP-myc cGAS GSAA/STING cells

(Fig. 5C).

We next carried out Southern blotting and its quantitative analysis to examine the

level of HBV DNA in HepG2.2.15 cGAS/STING cells. The results showed that the

level of intracellular HBV DNA was not reduced in HepG2.2.15 cGAS/STING cells,

compared with the control cells (Fig. 6A and the left panel of Fig. 6B). However, we

found that the infectivity of intracellular HBVcc was significantly reduced in

HepG2.2.15 cGAS/STING cells, compared with the control cells or HepG2.2.15 cGAS

GSAA/STING cells (the right panel of Fig. 6B). Moreover, we found that the

extracellular HBV DNA and the infectivity of extracellular HBVcc were reduced only

in HepG2.2.15 cGAS/STING cells (Fig. 6C). From these results, we conclude that the

cGAS-STING signaling pathway negatively regulates the viral assembly.

Low expression of endogenous cGAS in Li23 cells increased the permissiveness to

HBV infection

Our findings that HBV triggered an antiviral response through the cGAS-STING

signaling pathway suggested that the expression level of endogenous cGAS might affect

the permissiveness to HBV infection. To investigate this possibility, we first prepared

Li23/NTCP-myc cells stably expressing NTCP containing a myc tag, and then

established dozens of subcloned cell lines by the limited dilution of Li23/NTCP-myc

cells to obtain cell lines showing different levels of cGAS expression. Among the

obtained cell lines, we selected A7 cells, which showed the highest level of cGAS

expression, and A8 cells, which showed the lowest level of cGAS expression (Fig. 7A).

An HBV infection experiment using A7 and A8 cells in addition to parental

Li23/NTCP-myc cells revealed that the permissiveness to HBV infection in A7 cells

was lower than that in the parental cells and the permissiveness in A8 cells was higher

than that in the parental cells (Fig. 7B). At 13 days after HBVcc infection, we also

observed that ISG56 was induced in A7 cells, but not in A8 cells (Fig. 7C). In addition,

we observed that B34 and B48 subcloned cells, which showed a low level of cGAS

expression equivalent to that in A8 cells (Fig. S1A), also showed higher permissiveness

to HBV infection than that in the parental cells (Fig. S1B). These results also suggest

that cGAS is a host factor which may regulate the permissiveness to HBV infection.

Discussion

The DNA double helix is known to form a right-handed conformation (i.e., A-form or

B-form DNA) or left-handed conformation (i.e., Z-form DNA). Most DNA exists as the

B-form in vitro and in vivo. B-form DNA such as p-dAdT is recognized by DDX41 in

myeloid dendritic cells [22] (Fig. 7C). Interestingly, under certain conditions, DNA is

reported to transit from B-form to Z-form. As the case in vitro, it was previously

reported that the insertion of the sequence (dC-dG)n into plasmid DNA pBR322

promoted the transition from B-form DNA to Z-form DNA in their negatively

supercoiled cccDNA [23-25]. On the other hand, the antibodies specific to Z-form DNA

were previously shown to be present in the sera of patients with systemic lupus

erythematosus [26]. This result implies that Z-form DNA may be recognized in host

cells as “non-self” DNA, although the biological functions of Z-form DNA have

remained uncertain. In the present study, using Li23 cells expressing cGAS, we

demonstrated that the cGAS-STING signaling pathway was required for the recognition

of the synthetic p-dGdC (Z-form), HBV, HSV-1- or VACV-derived dsDNA (Fig. 3B). In

contrast, in PH5CH8 cells, which do not express cGAS, only p-dAdT (B-form DNA)

induced the innate immune response, and p-dGdC (Table 1), HBV-, HSV-1- or VACV-

derived dsDNA (data not shown) was not absolutely recognized. These results suggest

that the synthetic HBV-derived dsDNA as well as HSV-1- or VACV-derived dsDNA

takes the conformation of Z-form in the cells. According to other groups, the

cGAS-STING signaling pathway was required for the recognition of several viruses,

such as HSV-1 [11], VACV [11], and HIV [12]. We also demonstrated that the

cGAS-STING signaling pathway was required for the recognition of HBV dsDNA, and

functioned as an antiviral mechanism through the induction of ISG56 (Figs. 4C and 4E).

These results imply that cGAS may play an important role in the recognition of Z-form

DNA, such as viral replication intermediates produced from DNA virus as “non-self”

DNA (Fig. 7D).

The cGAS-STING signaling pathway also suppressed the infectivity of intracellular

HBV without a reduction of intracellular HBV DNA (Fig. 6B). This result suggests that

the cGAS-STING signaling pathway may negatively regulate the viral assembly. The

5’-cap structure of HBV pgRNA is essential for viral encapsidation [27]. On the other

hand, ISG56 is known to inhibit the replication of some viruses by inhibiting viral

translation through binding to the 5’-cap structure of viral RNA [15]. Further analysis is

needed to clarify whether the cGAS-STING signaling pathway inhibits viral

encapsidation or viral translation through the binding of ISG56 to the 5’-cap structure of

HBV pgRNA.

Recently it was reported that both HBV and HCV were able to replicate and

proliferate in a newly developed hepatoma cell line, HLCZ01 [28]. This result implies

that the common important host factor or factors for the complete life cycles of both

HBV and HCV are present in hepatocyte. We previously found that human hepatoma

HuH-7-derived RSc cells and Li23-derived ORL8c cells showed higher permissiveness

of HCV than their parental HuH-7 and Li23 cells, respectively [13, 29]. In the present

study, we demonstrated that the high permissiveness to HBV infection was related to

lower cGAS expression using several cell lines subcloned from Li23/NTCP-myc cells

(Figs. 7A and 7B, and Fig. S1). In this context, interestingly, Li23-derived ORL8c cells

showed defective expression of cGAS and showed no responsiveness to p-dGdC (data

not shown). From these results, it is predicted that ORL8c cells possess an environment

that is advantageous to HBV multiplication. ORL8c may be a useful cell line for the

development of both HBV and HCV-replicating cells.

Experimental procedures

Cell cultures and reagents

Human hepatoma Li23 cells [13] were cultured in modified medium for human

immortalized hepatocyte PH5CH8 cells [30] as previously described [13]. Other human

immortalized hepatocytes, NKNT-3 [31] and OUMS-29 cells [32], which were kindly

provided by Drs. Noriyuki Kobayashi and Masayoshi Namba (Okayama University),

were also maintained in this modified medium for human immortalized hepatocytes.

Human hepatoma HuH-7, PLC/PRF/5, HT17, HLE, and HepG2 cells were cultured in

Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented

with 10% fetal bovine serum as previously described [16, 18]. Li23 NS3/4A cells were

maintained in medium including blasticidin as previously described [19, 33].

HepG2.2.15 cells [14] were kindly provided by Dr. Takaji Wakita (National Institute of

Infectious Disease, Tokyo, Japan).

HepG2/NTCP-myc cells and Li23/NTCP-myc cells were prepared by the retroviral

transfer of myc-tagged NTCP. Limited dilution of Li23/NTCP-myc cells were

performed to establish the subcloned cell lines.

In vitro synthesized ligands such as p-IC, 5’-ppp dsRNA, p-dGdC, and p-dAdT

were purchased from InvivoGen (San Diego, CA, USA). Synthetic dsDNA derived

from HSV-1 and VACV were also purchased from InvivoGen. In vitro synthesized

p-AdT was purchased from The Midland Certified Reagent Company (Midland, TX,

USA).

Quantitative RT-PCR analysis

In vitro synthesized ligands were complexed with Lipofectamine 2000 (Invitrogen) for

the transfection as previously described [19]. Total cellular RNA was isolated from the

cells at 6 h after transfection by using an RNeasy Mini Kit (Qiagen, Hilden, Germany).

RT was performed as previously described [33]. For quantitative PCR, we used a SYBR

Premix Ex Taq Kit (TaKaRa Bio, Otsu, Japan) and the following primer sets: for ISG56

[34], IRF-3 [19], and GAPDH [35]. We also prepared the following forward and reverse

primer sets for cGAS, 5’-AAGCTCCGGGCGGTTTTGGA-3’ (forward) and

5’-AGGTGCAGAAATCTTCACGTGCTC-3’ (reverse); for STING,

5’-GTGGCTTGAGGGGAACCCGC-3’ (forward) and

5’-GGCTGGAGTGGGGCATCTTCT-3’ (reverse). These expression levels were

normalized to the levels of GAPDH mRNA. We used total RNA isolated from the cells

without the transfection of ligand as a control; (-). Data are the means ± SD from three

independent experiments.

RNA interference

Small interfering RNAs (siRNAs) targeting cGAS (Thermo Fisher Scientific, Waltham,

MA; M-015607-01-0005), STING (M-024333-00-0005), IRF-3 (M-006875-01-0005),

or nontargeting siRNAs (D-001206-13-20) were introduced into Li23 cells by

DharmaFECT transfection reagent (Thermo Fisher Scientific). The effects of siRNAs

were confirmed by the quantitative RT-PCR and Western blot analysis using anti-cGAS

(Cell Signaling Technology, Beverly, MA, USA), anti-STING (Cell Signaling

Technology) and anti-IRF-3 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies. At

3 days after the introduction of siRNAs, the knockdown cells were transfected with

several ligands.

Synthetic HBV dsDNA

Two oligonucleotides (nt 1750-1795 according to C_JPNAT clone with accession no.

AB246345) [36] derived from the positive- or negative-stranded HBV DNA were

synthesized in vitro. These oligonucleotides were annealed by heating at 70 degrees for

10 minutes followed by slow cooling to room temperature. The annealed product was

confirmed by agarose gel electrophoresis.

Generation of cells stably expressing exogenous cGAS or STING

To construct pCX4bsr/HA-cGAS and pCX4pur/Myc-STING retroviral vectors, we

introduced cGAS (accession no. NM_138441) and STING (accession no. NM_198282)

cDNA containing a full-length ORF into the pCX4bsr/HA or pCX4pur/Myc retroviral

vector, respectively, as previously reported [37]. These vectors were simultaneously

introduced into HepG2.2.15 cells, HepG2 cells or HepG2/NTCP-myc cells by the

retroviral transfer and subsequently selected the cells stably expressing exogenous

cGAS and STING by blasticidin and puromycin. We also introduced both pCX4bsr and

pCX4pur vectors into cells as control cells. At 3 days after transfection, total cellular

RNA, and the cell lysate were prepared from both these cells. The exogenous

expression of cGAS and STING were confirmed by the quantitative RT-PCR or Western

blot analysis using anti-HA (Cell Signaling Technology, Beverly, MA, USA) antibodies

as previously described [34].

HBV infection

HBVcc was prepared from the supernatant of HepG2.2.15 cells. The supernatant was

concentrated by the addition of one-fourth volume of 40% (w/v) polyethylene glycol

(PEG) 8000 in PBS (final 8% (w/v)) followed by incubation at 4 degrees overnight. The

precipitates, HBVcc, were collected by centrifugation and then used as the HBV

inoculum. Inoculation using HBVcc was performed with 1000 HBV genome

equivalents per cell in culture medium containing 4% PEG 8000 and 2% DMSO for 24

hr. At 24 hr after the inoculation, the culture medium was replaced with fresh medium.

Analysis of HBV DNA

For analysis of the intracellular HBV DNA, total cellular DNA was isolated by

UltraPure Phenol: Chloroform: Isoamyl Alcohol (25: 24: 1, v/v) (Invitrogen), and was

subjected to Southern blot analysis. Briefly, total cellular DNA was separated on 0.8%

agarose gel and then transferred to a Hybond-N+ membrane (GE Healthcare) using a

standard neutral transfer procedure. To prepare the DIG-labeled HBV minus-stranded

specific riboprobe, we constructed pTZ19R 1403-2907AS by introducing the antisense

strand of the HBV DNA fragment (nts 1403-2907 according to the D_IND60 clone with

accession no. AB246347, [36]) into pTZ19R. pTZ19R 1403-2907AS was linearized by

EcoRI to provide a template for synthesis of the DIG-labeled HBV minus-stranded

specific riboprobe using a T7 Megascript kit (Ambion, Austin, TX) and DIG RNA

labeling mix (Roche). The transferred membrane was hybridized with the DIG-labeled

HBV minus-stranded specific riboprobe, and then HBV DNA was detected using

anti-DIG antibody (Roche).

For analysis of the extracellular HBV DNA and viral infectivity, the supernatants of

HepG2-derived cells were collected. Extracellular DNA was isolated from the

supernatant by using a DNeasy Blood & Tissue Kit (Qiagen). Quantitative PCR analysis

of HBV DNA was performed as previously reported [21]. pC_JPNAT plasmid DNA

[36] was used as a standard to calculate the amount of HBV DNA. Data are the means ±

SD from three independent experiments.

The supernatant of HepG2-derived cells was also used for the inoculation to

HepG2/NTCPmyc cells in culture medium containing 4% PEG 8000 and 2% DMSO.

At 4 days after the inoculation, total RNA was isolated by using an RNeasy Mini Kit

(Qiagen). Quantitative RT-PCR analysis of HBV RNA was performed to evaluate the

viral infectivity of the supernatant as described below.

Analysis of HBV RNA

For the analysis of the intracellular HBV RNA, total RNA was isolated by using an

RNeasy Mini Kit (Qiagen), and was subjected to Northern blot analysis. Total RNA was

separated on 0.8% agarose gel and then transferred to a Hybond-N+ membrane (GE

Healthcare) using a standard transfer procedure. DIG-labeled HBV plus-stranded

specific riboprobe was prepared from pTZ19R 1426-1896 (corresponding to the sense

strand of HBV DNA; nts 1426-1896 of the D_IND60 clone) using a T7 Megascript kit

(Ambion, Austin, TX) and DIG RNA labeling mix (Roche). The transferred membrane

was hybridized with the DIG-labeled HBV plus-stranded specific riboprobe, and then

HBV RNA was detected using anti-DIG antibody (Roche).

Quantitative RT-PCR analysis of HBV RNA was performed as previously reported

[21]. pC_JPNAT plasmid DNA [36] was used as a standard to calculate the amount of

HBV RNA. Data are the means ± SD from three independent experiments.

Statistical analysis

The significance of differences among groups was determined using Student’s t-test.

P<0.05 was considered statistically significant.

Acknowledgments

We thank Marie Iwado, Yoshiko Ueeda, Masayo Takemoto, and Takashi Nakamura

for their technical assistance. This work was supported by Grants-in Aid for Practical

Research on hepatitis from the Ministry of Health, Labor, and Welfare of Japan; and by

JSPS KAKENHI Grant Number 25293110.

Author contributions

HD and NK designed the experiments. HD performed most of the experiments. NO

contributed Li23/NTCP-myc cells. YU and NK contributed the limited dilution of

Li23/NTCP-myc cells. MS and MM contributed HBV plasmids. HD, NO, SS, MI and

NK analyzed the data. HD and NK wrote the paper.

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Table 1

Responsibility against ligands in various human hepatocyte cells

Ligand

dsRNA dsDNA RNA:DNA duplex

p-IC 5’-ppp dsRNA

p-dGdC p-dAdT p-AdT

Human immortalized hepatocyte cell

PH5CH8 1866 196 9 2147 5

NKNT-3 206 41 396 1948 9

OUMS29 481 85 8 2097 7

Human hepatoma cell Li23 2109 7 102 655 3

HuH-7 1281 13 7 34 5

HepG2 6466 11 6 1140 5

PLC/PRF/5 461 11 9 75 7

HT17 1462 6 13 206 3

HLE 224 9 9 55 5

Quantitative RT-PCR analysis of ISG56 mRNA was performed. Each level of ISG56 mRNA

level was calculated relative to the level in the cells without treatment of the ligand, which was

set at 10.

Figure Legends

Fig. 1. cGAS was required for the p-dGdC-triggered signaling pathway in Li23 cells.

(A) Quantitative RT-PCR analysis of ISG56 mRNA in Li23 NS3/4A cells treated with

p-IC (left panel), p-dAdT (central panel), or p-dGdC (right panel). The level of ISG56

mRNA was calculated relative to the level in Li23 Cont cells without ligand treatment;

(-), which was set at 1. (B) Quantitative RT-PCR analysis of cGAS mRNA in several

human cell lines. Total RNA derived from human normal liver was used as the control.

The level of cGAS mRNA was calculated relative to the level in Li23 cells, which was

set at 100%. (C) (Left panel) Quantitative RT-PCR analysis of cGAS mRNA in Li23

cells transfected with cGAS-specific (designated Li23 sicGAS) or control (designated

Li23 siCont) siRNA. The level of cGAS mRNA was calculated relative to the level in

Li23 siCont cells, which was assigned as 100%. (Right panel) Western blot analysis of

cGAS, STING, and IRF-3 in Li23 sicGAS cells. -actin was included as a loading

control. (D) (Upper panels) Quantitative RT-PCR analysis of ISG56 mRNA in Li23

sicGAS cells treated with p-IC (left panel), p-dAdT (central panel) or p-dGdC (right

panel). The level of ISG56 mRNA was calculated relative to the level in Li23 siCont

cells without ligand treatment; (-), which was set at 1. (Lower panels) Western blot

analysis of ISG56 in Li23 sicGAS cells treated with p-IC (left panel), p-dAdT (central

panel) or p-dGdC (right panel).

Fig. 2. STING and IRF-3 were required for the p-dGdC-triggered signaling pathway in

Li23 cells. (A) (Upper panel) Quantitative RT-PCR analysis of STING mRNA in Li23

cells transfected with STING-specific (designated Li23 siSTING) or control (designated

Li23 siCont) siRNA. The level of STING mRNA was calculated relative to the level in

Li23 siCont cells, which was assigned as 100%. (Lower panel) Western blot analysis of

STING in Li23 siSTING cells. (B) (Upper panel) Quantitative RT-PCR analysis of

IRF-3 mRNA in Li23 cells transfected with IRF-3-specific (designated Li23 siIRF-3) or

control (designated Li23 siCont) siRNA. The level of IRF-3 mRNA was calculated

relative to the level in Li23 siCont cells, which was assigned as 100%. (Lower panel)

Western blot analysis of IRF-3 in Li23 siIRF-3 cells. (C) (Upper panels) Quantitative

RT-PCR analysis of ISG56 mRNA in Li23 siSTING cells treated with p-dGdC (left

panel) and p-dAdT (right panel). The level of ISG56 mRNA was calculated relative to

the level in Li23 siCont cells without ligand treatment; (-), which was set at 1. (Lower

panels) Western blot analysis of ISG56 in Li23 siSTING cells treated with p-dGdC (left

panel) and p-dAdT (right panel). (D) (Upper panels) Quantitative RT-PCR analysis of

ISG56 mRNA in Li23 siIRF-3 cells treated with p-dGdC (left panel) and p-dAdT (right

panel). The level of ISG56 mRNA was calculated relative to the level in Li23 siCont

cells without ligand treatment; (-), which was set at 1. (Lower panels) Western blot

analysis of ISG56 in Li23 siIRF-3 cells treated with p-dGdC (left panel) and p-dAdT

(right panel).

Fig. 3. HBV-derived dsDNA was recognized through the cGAS-STING signaling

pathway in Li23 cells. (A) Agarose gel electrophoresis of the synthesized HBV-derived

dsDNA. P-dAdT, p-dGdC, VACV-derived dsDNA, and HSV-derived dsDNA were also

loaded on agarose gel (2.0%) for reference. (B) Quantitative RT-PCR analysis of ISG56

mRNA in Li23 sicGAS cells (left panel) or Li23 siSTING cells (right panel) treated

with p-dGdC, HBV-, VACV-, or HSV-derived dsDNA. The level of ISG56 mRNA was

calculated relative to the level in Li23 siCont cells without ligand treatment; (-), which

was set at 1. (C) Quantitative RT-PCR analysis of ISG56 mRNA in Li23 cells treated

with HBV positive- or negative-stranded ssDNA (designated Pos or Neg, respectively).

The level of ISG56 mRNA was calculated relative to the level in Li23 cells without

ligand treatment (-), which was set at 1.

Fig. 4. HBV triggered the cGAS-STING signaling pathway in HepG2.2.15 cells. (A)

(Upper panels) Quantitative RT-PCR analysis of cGAS (left panel), STING (central

panel), and IRF-3 (right panel) mRNAs in HepG2 cells or HepG2.2.15 cells. Each

mRNA level was calculated relative to the level in Li23 cells, which was assigned a

value of 100%. (Lower panel) Western blot analysis of cGAS in HepG2 cells or

HepG2.2.15 cells. (B) Quantitative RT-PCR analysis of cGAS mRNAs in HepG2

cGAS/STING cells or HepG2.2.15 cGAS/STING cells. HepG2 Cont cells, HepG2

cGAS GSAA/STING cells, HepG2.2.15 Cont cells and HepG2.2.15 cGAS

GSAA/STING cells were used as control cells. Each mRNA level was calculated

relative to the level in HepG2 cGAS/STING cells, which was assigned a value of 100%.

(C) Quantitative RT-PCR analysis of ISG56 mRNAs in HepG2 cGAS/STING cells or

HepG2.2.15 cGAS/STING cells. Control cells were used as described in Fig. 4B. Each

mRNA level was calculated relative to the level in HepG2 Cont cells, which was set at 1.

(D) Western blot analysis of ISG15 in HepG2 cGAS/STING cells or HepG2.2.15

cGAS/STING cells. Control cells were used as described in Fig. 4B. β-actin was

included as a loading control. (E) Quantitative RT-PCR analysis of ISG56 mRNAs in

HBV-infected HepG2/NTCP-myc cGAS/STING cells. HepG2/NTCP-myc Cont cells

and HepG2/NTCP-myc cGAS GSAA/STING cells were used as control cells. The

supernatant of HepG2.2.15 was used as HBVcc. Each mRNA level was calculated

relative to the level in mock-infected HepG2/NTCP-myc Cont cells, which was set at 1.

Fig. 5. The cGAS-STING signaling pathway significantly reduced the levels of HBV

RNA in HepG2.2.15 cells. (A) Northern blot analysis of the total amount of HBV

transcript in HepG2.2.15 cGAS/STING cells. HepG2.2.15 Cont cells and HepG2.2.15

cGAS GSAA/STING cells were used as control cells. β-actin was included as a

loading control. (B) Quantitative RT-PCR analysis of the total HBV transcript and

pgRNA in HepG2.2.15 cGAS/STING cells. Control cells were used as described in Fig.

5A. Total RNA extracted from the cells was subjected to quantitative RT-PCR analysis.

(C) Quantitative RT-PCR analysis of the total HBV transcript in HBV-infected

HepG2/NTCP-myc cGAS/STING cells. Control cells and HBVcc were used as

described in Fig. 4E. Total RNA extracted from the cells was subjected to quantitative

RT-PCR analysis.

Fig. 6. The cGAS-STING signaling pathway showed an antiviral response towards

HBV through the suppression of viral assembly. (A) Southern blot analysis of HBV

DNA in HepG2.2.15 Cont, cGAS/STING, or cGAS GSAA/STING cells. M:

genome-length HBV DNA (pC_JPNAT plasmid DNA cleaved by EcoRI and HindIII);

RC: HBV relaxed circular DNA; SS: HBV ssDNA. (B) Quantitative PCR analysis of

the total amount of HBV DNA in HepG2.2.15 Cont, cGAS/STING or cGAS

GSAA/STING cells (left panel). Total cellular DNA extracted from the cells was

subjected to quantitative PCR analysis. Quantitative RT-PCR analysis of the total HBV

transcript in HepG2/NTCP-myc cells was performed at 5 days after infection with

intracellular HBV (right panel). The lysate was recovered as intracellular HBV from

HepG2.2.15 Cont, cGAS/STING or cGAS GSAA/STING cells. Total RNA extracted

from intracellular HBV-infected cells was subjected to quantitative RT-PCR analysis.

(C) Quantitative PCR analysis of HBV total DNA in the supernatant released from

HepG2.2.15 Cont, cGAS/STING, or cGAS GSAA/STING cells (left panel). Total DNA

extracted from the supernatant was subjected to quantitative PCR analysis. Quantitative

RT-PCR analysis of the total HBV transcript in HepG2/NTCP-myc cells at 5 days after

infection with extracellular HBV (right panel). The supernatant was recovered as

extracellular HBV from HepG2.2.15 Cont, cGAS/STING, or cGAS GSAA/STING cells.

Total RNA extracted from extracellular HBV-infected cells was subjected to

quantitative RT-PCR analysis.

Fig. 7. The expression level of endogenous cGAS was inversely correlated with the

permissiveness to HBV infection. (A) Quantitative RT-PCR analysis of cGAS mRNAs

in Li23/NTCP-myc cells (Parent), and their subcloned A7, or A8 cells. Each mRNA

level was calculated relative to the level in parental Li23/NTCP-myc cells, which was

assigned a value of 100%. (B) Quantitative RT-PCR analysis of the total amount of

HBV transcript in HBV-infected Li23/NTCP-myc Parent, A7 or A8 cells. HBVcc were

used as described in Fig. 4E. Total RNA extracted from the cells was subjected to

quantitative RT-PCR analysis. ND, not determined. (C) Quantitative RT-PCR analysis

of ISG56 mRNAs in HBV-infected Li23/NTCP-myc Parent, A7, or A8 cells. The

supernatant of HepG2.2.15 was used as HBVcc. Each mRNA level was calculated

relative to the level in mock-infected cells, which was set at 1. (D) Proposed models of

the cGAS-STING signaling pathways triggered by HBV.

Supporting material

Fig. S1. The subcloned cell lines of Li23/NTCP-myc cells, in which the cGAS

expression was at the same low level as in A8 cells, also showed higher permissiveness

to HBV infection than that in the parental cells. (A) Quantitative RT-PCR analysis of

cGAS mRNAs in Li23/NTCP-myc cells (Parent), and their subcloned A8, B34, or B48

cells. Each mRNA level was calculated relative to the level in parental Li23/NTCP-myc

cells, which was assigned a value of 100%. (B) Quantitative RT-PCR analysis of the

total amount of HBV transcript in HBV-infected Li23/NTCP-myc Parent, A8, B34, or

B48 cells. HBVcc were used as described in Fig. 4E. Total RNA extracted from the cells

was subjected to quantitative RT-PCR analysis.

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