JVI Accepts, published online ahead of print on 10 December 2014 J. Virol. doi:10.1128/JVI.02760-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Hepatitis B virus polymerase disrupts K63-linked ubiquitination of STING to block

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innate cytosolic DNA-sensing pathways

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Yinghui Liu1*, Jianhua Li2*, Jieliang Chen2,Yaming Li2, Weixia Wang2, Xiaoting

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Du2, Wuhui Song2, Wen Zhang2, Li Lin2, Zhenghong Yuan1,2#

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Institutes of Biomedical Sciences, Fudan University, Shanghai, PR China1; Key

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Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan

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University, Shanghai, PR China2.

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Running Head: HBV polymerase blocks STING-mediated IFN induction

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# Address correspondence to Zhenghong Yuan, [email protected].

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*Y. L. and J. L. contributed equally to this work.

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The word count for the abstract: 388 (238 in Abstact, 150 in Importance)

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The word count for the text: 6337

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Abstract

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The cellular innate immune system recognizing pathogen infection is essential for

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host defense against viruses. In parallel, viruses have developed a variety of strategies

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to evade the innate immunity. The hepatitis B virus (HBV), a DNA virus that causes

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chronic hepatitis, has been shown to inhibit the RNA helicase RIG-I-mediated

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interferon (IFN) induction. However, it is still unknown whether HBV could affect

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the host DNA-sensing pathways. Here we report that in transient HBV-transfected

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Huh7 cells, HBV stably producing cell line HepAD38, and HBV-infected HepaRG

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cells and primary human hepatocytes, HBV markedly interfered with IFN-β induction

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and antiviral immunity mediated by STING, which has been identified as a central

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factor in foreign DNA recognition and antiviral innate immunity. Screening analysis

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demonstrated that the viral polymerase (Pol), but not other HBV-encoded proteins,

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was able to inhibit STING-stimulated interferon regulatory factor 3 (IRF3) activation

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and IFN-β induction. Moreover, the reverse transcriptase (RT) and the ribonuclease H

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(RH) domains of Pol were identified to be responsible for the inhibitory effects.

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Furthermore, Pol was shown to physically associate with STING and dramatically

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decrease the K63-linked polyubiquitination of STING via its RT domain without

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altering the expression level of STING. Taken together, these observations suggest

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that besides its inherent catalytic function, Pol has a role in suppression of IFN-β

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production by direct interaction with STING and subsequent disruption of its

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K63-linked ubiquitination, providing a new mechanism for HBV to counteract the

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innate DNA-sensing pathways.

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Importance

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Although whether and how HBV infection induces the innate immune responses is

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still controversial, it becomes increasingly clear that HBV has developed strategies to

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counteract the pattern recognition receptors-mediated signaling pathways. Previous

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studies have shown that type I IFN induction activated by the host RNA sensors could

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be inhibited by HBV. However, it remains unknown whether HBV as a DNA virus

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utilizes evasion mechanisms against the foreign DNA-elicited antiviral signalings. In

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recent years, the cytosolic DNA sensor and key adaptor STING has been 2 / 32

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demonstrated to be essential in multiple foreign DNA-elicited innate immune

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signalings. Here, for the first time, we reported STING as a new target of HBV to

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antagonize the IFN induction and identified the viral polymerase responsible for the

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inhibitory effect, thus providing an additional molecular mechanism by which HBV

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evades the innate immunity and implying HBV polymerase is a multifunctional

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immunomodulatory protein besides its inherent catalytic function.

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Introduction

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Hepatitis B virus（HBV） is one of the most important pathogens causing liver

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diseases. Worldwide, approximately 350-400 million individuals are chronically

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infected, many of whom are at increased risk of developing cirrhosis and

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hepatocellular carcinoma (HCC)(1, 2). Although the underlying mechanisms leading

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to chronic HBV infection remain to be clearly defined, the outcome of HBV infection

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is thought to be the results of complex interactions between replicating HBV and the

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host immune system (3).

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The innate immunity constitutes the first line of defense against invading pathogens,

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which recognizes the pathogen-associated molecular patterns (PAMPs) through

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germline-encoded pattern recognition receptors (PRRs). Viral infection usually

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activates some PRR(s), leading to type I interferons (IFNs, including IFN-α and

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IFN-β) and inflammatory activities (4, 5). However, viruses including HBV have

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developed a variety of strategies to counteract the host immune responses for the

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benefit of their survival. It is reported that HBV surface antigen (HBs), e antigen

83

(HBeAg) and HBV virions could inhibit Toll-like receptors (TLRs)-mediated

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production of type I IFN and pro-inflammatory cytokines in murine liver cells (6). In

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addition, HBV x protein (HBx) was reported to negatively regulate retinoic

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acid-inducible gene I (RIG-I)-mediated antiviral responses (7-9), while the viral

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polymerase (Pol) was shown to suppress type I IFN induction through impairing

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RIG-I- and TLR3-stimulated signalings (10, 11), both of which are RNA-sensing

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pathways. Considering that HBV is a DNA-containing virus with a genome size of

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3.2 Kb and there are at least two types of viral DNAs distinct from the host DNA 3 / 32

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including relaxed circular DNA (rcDNA) and covalently closed circular DNA

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(cccDNA) during its life cycle, we thus speculate that HBV may also have strategies

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to interfere with the host DNA-sensing pathways.

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Significant progress has been made in recent years in understanding how the innate

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immune system detects non-self DNA molecules or DNA-containing pathogens.

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Several proteins, including DNA-dependent activator of IFN-regulatory factors

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(DAI)(12, 13), absent in melanoma 2 (AIM2) (14-16), the member of the PYHIN

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protein family IFI16 (17), the member of the DEXDc family of helicases DDX41 (18),

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the cGAMP synthase (cGAS) (19, 20) have been identified as DNA sensors.

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Interestingly, the downstream signalings activated by most of these DNA sensors

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converge on an essential signal transducer, the stimulator of interferon genes (STING,

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also known as MITA, ERIS, TMEM173 and MPYS)(21-24). Meanwhile, STING is

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reported as a direct innate immune sensor of cyclic diguanylate monophosphate

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(c-di-GMP), a bacterial second messenger(25). Collectively, STING, functioning at

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the signaling “traffic junction”, plays a critical role in the regulation of the immune

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response to microbial nucleic acids, particularly the cytosolic DNA and cyclic

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dinucleotides (CDNs). However, little is known about whether and how HBV disturbs

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STING signaling in human hepatocytes.

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Therefore, the aim of this study was to investigate the possible impact of HBV

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replication on the STING-mediated type I IFN induction pathway. The results

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revealed a novel mechanism employed by HBV to escape the innate immunity and

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provided evidence for a new role of the viral polymerase in the inhibition of IFN-β

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production through disrupting the K63-linked ubiquitination of STING.

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Materials and Methods

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Plasmids and viruses

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The following expression plasmids were generously provided: pIFN-β-Luc (Rongtuan

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Lin, McGill University, Canada); pEF-STING, and p-55C1B-Luc (Takashi Fujita,

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Kyoto University, Japan); pFlag-MITA, pcDNA3.1-HA-MITA, pFlag-TRIM32 and

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pFlag-TRIM56

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pcDNA3.1-CMV-HBV1.1 (Youhua Xie, Fudan University, China) which contains the

(Hongbing

Shu, 4 / 32

Wuhan

University,

China);

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wild-type HBV 1.1-mer overlength genomic sequence. pHBV1.3, pFlag-Pol

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(pcDNA3.1-Flag-Pol and pQCXIP-Flag-Pol), the series of truncated mutants of Pol,

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pcDNA3.1-Flag-Core,

pcDNA3.1-Flag-HBx,

pcDNA3.1-Flag-Precore,

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pcDNA3.1-Flag-HBsAg,

pFlag-TBK1,pHA-RIG-I,

pCMV-Myc-Pol

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pcDNA3-HA-Ub were described previously (10, 26-29). pHBV1.3-ΔPol and

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pCMV-HBV-ΔPol, defective in viral polymerase expression, were derived from

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pHBV1.3 and pcDNA3.1-CMV-HBV1.1 respectively by introducing a frameshift

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mutation into the Pol gene after codon 108. The mutation strategy was described

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previously(30). pHBV1.3-YMHD and pFlag-Pol-YMHD, lacking the polymerase

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catalytic active site, were derived from pHBV1.3 and pFlag-Pol, respectively by

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introducing a substitution mutation into the Pol gene. The amino acid was exchanged

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from

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GGCTTTCAGTTATATGCATGATGTGGT (the mutated nucleotide was underlined).

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The mutation strategy was described previously (31). The carboxy-terminally

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Flag-tagged Pol pPol-Flag was constructed by inserting a DNA fragment encoding

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three repeats of the flag epitope tag at the carboxy terminus of Pol on the pcDNA3.1

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vector.

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The mutants pHA-Ub-K48R and pHA-Ub-K63 were derived from pcDNA3-HA-Ub.

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In mutant pHA-Ub-K48R, the lysine at position 48 was substituted with arginine by in

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vitro mutagenesis. In pHA-Ub-K63, all lysines except that at position63 (including

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the lysines at positions 6, 11, 27, 29, 33 and 48) were mutated to arginines. pRL-TK

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was purchased from Promega. NDV-GFP (a gift from Dr Yan Yuan, University of

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Pennsylvania,

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specific-pathogen-free-maintained chicken eggs.

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Antibodies and reagents

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Anti-β-actin, anti-Flag M2, anti-GFP and anti-HA antibodies were purchased from

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Sigma-Aldrich. Anti-c-Myc and anti-IRF3 (FL425) antibodies were obtained from

148

Santa Cruz Biotechnology. Anti-STING antibody was obtained from Proteintech.

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Anti-Core, anti-HBs and anti-Ub antibodies were purchased from Dako, Shanghai

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Long Island Biotech and Covance, respectively. Anti-TRIM32 antibody was a gift

YMDD

to

YMHD

PA,

in

USA)

the

polymerase

was

propagated

5 / 32

sequence.

and

The

and

primer

purified

is

from

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from Dr. Hongbing Shu (Wuhan University, China). The proteasome inhibitor

152

MG-132 was purchased from Calbiochem. cGAMP and polydA:dT were purchased

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from Biolog and Amersham Biosciences, respectively.

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Cell culture and transfection

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The cell lines HEK293, HEK293T, Vero(obtained from the Cell Bank of the Chinese

156

Academy of Science (Shanghai, China)) and Huh7 were cultured in Dulbecco’s

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modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS; Gibco),

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penicillin (100 IU ml-1; Gibco) and streptomycin(100 μg ml-1; Gibco), in a 5% CO2

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atmosphere at 37℃. The HepG2-derived HBV-producing stable cell line HepAD38

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which was a kind gift from Yumei Wen, was maintained as described previously (32).

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Additionally, HepAD38 cells were grown in presence or absence of 1 μg ml-1

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doxycycline (Dox) to regulate HBV pregenomic RNA transcription. PH5CH8, a

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simian virus 40 large T antigen-immortalized non-neoplastic human hepatic cell line

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with intact capacity for type I IFN induction was maintained as described previously

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(33). HepaRG cells (34) were purchased from BIOPREDIC INTERNATIONAL,

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France. To obtain the differentiated HepaRG (dHepaRG), the cells were cultured for

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two weeks in standard medium, then for two more weeks in medium supplemented

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with 1.8% dimethylsulfoxide according to the manufacturers’ instructions. Primary

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human hepatocytes (PHHs) were purchased from Shanghai RILD Inc. (Shanghai,

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China). The cell culture was performed as described previously with slight

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modification(35).The cell pellet containing PHHs was resuspended in the plating

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medium of Williams E medium supplemented with 10% FBS 5 μg ml-1 transferrin, 5

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ng ml-1 sodium selenite, 3μg ml-1 insulin, 2 mM L-glutamine, 100 U ml-1 penicillin,

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100 μg ml-1 streptomycin. The cells were plated in the collagen I pre-coated chambers

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(Lab-Tek) or the cell culture plates. 5 hr after plating, medium were changed to pri-

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mary hepatocytes maintenance medium (PMM), that is, Williams E medium

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supplemented with 5% FBS, 5 μg ml-1 transferrin, 10 ng ml-1 EGF, 3 μg ml-1 insulin, 2

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mM L-glutamine, 18 μg ml-1 hydrocortisone, 40 ng ml-1 dexamethasone, 5 ng ml-1

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sodium selenite, 2% DMSO, 100 U ml-1 penicillin,100 μg ml-1 streptomycin.

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Transient transfection was performed with the indicated plasmids using Fugene HP 6 / 32

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(Roche) or Lipofectamine 2000 (Invitrogen), according to the manufacturer’s

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instructions. Digitonin permeabilization was utilized to deliver cGAMP into cultured

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cells as described previously (36).

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HBV infection of HepaRGs and PHHs

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HBV infection of the hepatocytes was performed as described previously with slight

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modification (37). The dHepaRG cells or PHHs were incubated overnight with

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HBV-positive sera at approximate 100 genome equivalent copies of HBV per cell (1

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vol of infectious pooled serum from 10 CHB patients diluted in 10 vol of culture

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medium) containing 4% PEG 8000.After incubation, cells were rinsed three times.

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The HBeAg secretion and the viral DNA in the medium were determined every two

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or three days post-infection (data not shown). The cells successfully infected with

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HBV were processed to further treatment and experiments at least nine days

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post-infection.

194

The HBV-positive sera were collected from CHB patients in Shanghai Public Health

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Clinical Center with informed consent and the approval of the institutional ethics

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committee.

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Dual-luciferase reporter assay

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Cells (1×105) seeded in a 24-well plate were cultured overnight and then transfected

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with the indicated stimulator plasmid (such as pSTING), reporter plasmid (pRL-TK,

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pIFN-β-Luc or p-55C1B-Luc ) and the indicated plasmids using either Lipofectamine

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2000 (Invitrogen) or Fugene HP (Roche) according to the manufacturer’s instructions.

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At 36h post transfection, cells were lysed with passive lysis buffer and assayed using

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a Dual-Luciferase Assay kit (Promega). Data were processed as described previously

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(10). Data are expressed as the mean fold induction ± SD relative to control levels.

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The results are representative of three independent experiments, each performed in

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triplicate.

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Real-time RT-PCR

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Total cellular RNA was extracted with TRIzol reagent (Invitrogen), and then

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subjected to reverse-transcription using

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manufacturer’s instructions. The cDNA samples were subjected to real-time PCR 7 / 32

the

Kit

(TaKaRa)

following

the

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using primers specific for human IFN-β or human interferon stimulated gene

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56(ISG56) and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH): IFN-β

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forward,

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5′-CTTCAGGTAATGCAGAATCC-3′;

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5′-TAGCCAACATGTCCTCACAGAC-3′,and

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5′-TCTTCTACCACTGGTTTCATGC-3′;

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5′-GGTATCGTGGAAGGACTCATGA-3′,

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5′-ATGCCAGTGGCTTCCCGTTCAGC-3′. For comparisons, transcription of

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IFN-β and ISG56 was normalized to that of GAPDH. Data are expressed as the mean

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fold induction ± SD relative to control levels. The results are representative of three

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independent experiments, each performed in triplicate.

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IFN-β ELISA

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The level of IFN-β in the culture medium was measured using an ELISA kit for

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human IFN-β (PBL Interferon Source) according to the manufacturer’s instructions.

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Immunoprecipitation and Western blot analysis

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For immunoprecipitation, HEK293T (1×107) or HEK293 (4×107) cells were

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transfected with various combinations of plasmids using Lipofectamine 2000

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(Invitrogen) for 48 h and then lysed in immunoprecipitation buffer（50 mM Tris-HCl

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pH7.5, 150 mM NaCl, 0.5% (vol/vol) NP-40, 10% glycerol, 1mM EDTA）and

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supplemented with a protease inhibitor cocktail (Roche). Cell debris was removed by

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centrifugation at 12,000 g for 10 min at 4°C. The supernatants were collected,

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precleared with protein A/G Plus agarose beads (Santa Cruz) for 30min , and

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incubated with an anti-FLAG or anti-HA antibodies at 4°C. After 2 h, 25μl of 1:1

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slurry of protein A/G Plus agarose beads was added and incubated for additional 2h.

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The immunoprecipitates were washed five times with lysis buffer and boiled in 1%

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(wt/vol) SDS sample buffer, followed by SDS-PAGE and Western blot analysis as

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described previously (27).

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Confocal microscopy

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Immunofluorescence was performed as described (10). The fluorescence was

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observed under a confocal fluorescence microscope (Leica TCS SP2).

5′-GATTCATCTAGCACTGGCTGG-3′,and

8 / 32

ISG56

reverse, forward reverse,

GAPDH and

forward, reverse,

241

Southern blot analysis

242

Intracellular HBV core particle-associated DNA in Huh7 cells was extracted at 48h

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post-transfection and Southern blot analysis was performed as described previously

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(29).

245

Native PAGE

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For the detection of IRF3 dimerization, 3×105 HEK293 cells were seeded into a

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12-well plate, cultured overnight and then transfected with the indicated amounts of

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pEF-STING and pQCXIP-Flag-Pol (empty vector was used to balance the total

249

amount of DNA) using Lipofectamine 2000. After 24 h, cells were selected with

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puromycin (1.5μg ml-1) for 36 h. The cells were harvested with 40μl ice-cold lysis

251

buffer (50 mM Tris/HCl, (pH 7.5), 150 mM NaCl and 0.5% NP-40 containing 1×

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Roche protease inhibitors). After centrifugation at 13000 g for 10 min, supernatants

253

were quantified using a BCA assay (Thermo Scientific) and diluted with 5×native

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PAGE sample buffer (312.5 mM Tris/HCl (pH 6.8), 75% glycerol and 0.25%

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bromophenol blue), and then 60 μg of total protein was applied to a pre-runned 6%

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native gel for separation. After electrophoresis, the proteins were transferred onto a

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nitrocellulose membrane for Western blot.

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For the detection of HBV core capsids, cells were harvested with the above lysis

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buffer at 36h post-transfection and then centrifugated at 13000 g for 10 min. The

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quantified supernatants were subjected to a native agarose gel, blotted onto a

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nitrocellulose membrane, and detected for the formation of core capsids using the

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anti-Core antibody.

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In vivo ubiquitination assay

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HEK293T cells in a 60-mm-diameter dish were co-transfected with the indicated

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plasmids using Lipofectamine 2000 (Invitrogen). For the detection of STING

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ubiquitination, at 36 h post transfection, cells were treated with the proteasome

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inhibitor MG-132 at a final concentration of 20 μM for 6 h.The cells were washed

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twice with PBS and lysed with ice-cold RIPA buffer (50 mM Tris-HCl (pH 7.5),

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150mM NaCl, 1%NP-40, 0.5% Deoxycholic acid sodium, 0.1% SDS, supplemented

270

with a protease inhibitor cocktail (Roche) and 20μM MG132) for 5-10 min. Cell 9 / 32

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debris was removed by centrifugation at 12,000 g for 10 min at 4°C. The supernatants

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were divided into two aliquots. One aliquot (5%) was prepared for Western blot. The

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second aliquot (95%) was sonicated five times to shear DNA. The soluble lysates

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were then immunoprecipitated with anti-HA antibody followed by three washes with

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RIPA buffer. HA-tagged proteins were resolved by SDS-PAGE and sequentially

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blotted with the indicated antibodies.

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Infection protection assay

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Huh7 cells were seeded in a 24-well plate and transfected with the indicated plasmids.

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After 36h, the cells were mock-treated or treated with 100nM cGAMP for 16h. The

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supernatants were collected and filtered by 0.22μm filters (Millipore). Vero cells in

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24-well plate were incubated with the filtered Huh7 supernatants overnight, and then

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challenged with 80 haemagglutinin units (HAU) ml-1 of NDV-GFP for additional 24h,

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followed by observation under fluorescent microscope and analysis by Western blot.

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Statistical analysis

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Data were analyzed using Student’s t-test and presented as the mean ± Standard

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deviations. A value of P﹤0.05 was considered to be statistically significant.

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Results

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1. HBV inhibits STING-mediated IFN-β induction and antiviral immune

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response.

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To explore whether HBV could regulate STING-mediated type I IFN induction, we

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first test the overexpressed STING-stimulated IFN-β promoter activation inHuh7 cells

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transfected with two kinds of HBV replicative constructs, pHBV1.3 or pCMV-HBV

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in which HBV replication is driven by either HBV native promoter or the more potent

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cytomegalovirus (CMV) promoter, respectively. Meanwhile, we monitored HBV

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replication level by detecting the formation of core capsids using native agarose gel.

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As shown in Fig. 1A (left part), STING-mediated IFN-β promoter activation was

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abrogated in a dose-dependent manner in both of the HBV replicative

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plasmid-transfected cells, particularly in the cells transfected with pCMV-HBV in

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which higher level of core capsids was detected (Fig.1A, right part). The interferon

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regulatory factor 3 (IRF3), as a key transcriptional factor, is essential for 10 / 32

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STING-stimulated signaling transduction and IFN-β induction (22, 24). To further

302

confirm the effect of HBV on STING-mediated IFN-β induction, the effect of HBV

303

on STING-mediated IRF3 activation was tested using the reporter plasmid

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p-55C1B-Luc in which the luciferase expression was driven by a promoter containing

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repeated IRF-binding sites. The results showed that HBV effectively inhibited the

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STING-mediated IRF3 activation (Fig. 1B).

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Cyclic GMP-AMP (cGAMP) has been identified as a specific upstream activator of

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STING to induce type I IFN production (19, 20, 38, 39). Because STING expression

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was barely detectable in Huh7 cells and HepaAD38 cells (Fig. 1C) as reported

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previously (40), we co-transfected the cells with STING-encoding plasmids to

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reconstitute the responsiveness of the cells to cGAMP when reporter plasmids and

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plasmids containing HBV genome or control vectors were transfected into the cells.

313

Upon cGAMP stimulation, significant activation of IFN-β promoter was observed,

314

however, it was markedly impaired in the cells transfected with pHBV1.3 or

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pCMV-HBV (Fig.1D).Moreover, the amount of cGAMP-triggered IFN- production

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in Huh7 cells was also decreased in the presence of HBV replication in a

317

dose-dependent manner (Fig. 1E), indicating that the cGAMP-STING-mediated

318

signaling pathway was suppressed by HBV.

319

To further evaluate the inhibitory effect of HBV on the STING-mediated antiviral

320

response, we performed a virus infection protection assay, as described previously

321

(10). For this, the antiviral activities of supernatants harvested from IFN-α-or

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cGAMP-treated Huh7 cells with or without HBV replication were assayed in Vero

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cells challenged with the Newcastle disease viral particles tagged with green

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fluorescent protein (NDV-GFP) by determination of the expression level of extent of

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green fluorescence. The results showed that NDV-GFP expression was diminished in

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Vero cells pre-treated with the supernatants from cGAMP- and IFN-α-treated Huh7

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cells (Fig. 1F lanes/panels 4 and 8). In contrast, the supernatants from Huh7 cells

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transfected with the pHBV1.3construct lost antiviral activities depending on the

329

transfected dose, as judged by dim green fluorescence (Fig. 1F lanes/panels 5,6 and 7),

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implying that the innate antiviral response induced by cGAMP in Huh7 cells was 11 / 32

331

remarkably decreased in the presence of HBV.

332

To better understand the interference of HBV on STING-induced type I IFN

333

production in the HBV stably producing cell model, we tested the cGAMP-stimulated

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IFN-β promoter activation in HepaAD38 cell line, which expresses HBV under the

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control of an inducible tetracycline-off (Tet-off) promoter (32). The cells were

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pre-transfected with the STING expressing plasmid before cGAMP stimulation for

337

the reasons mentioned above that the endogenous expression level of STING is rather

338

low in HepAD38 cells. As shown in Fig. 1G, both 100nM and 400nM of

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cGAMP-stimulated IFN-β promoter activation was statistically lower, almost 35% in

340

the cells without doxycycline than those with doxycycline.

341

To further validate whether the suppression of HBV on STING signaling occuring

342

a natural infection of hepatocytes, differentiated HepaRG (dHepaRG) cells and PHHs

343

infected with HBV-positive sera were mock-treated or treated with cGAMP. In

344

dHepaRG cells, the immunofluorescece results showed that the IRF3 protein was

345

predominantly localized in the cytoplasm in the mock cells. Upon cGAMP

346

stimulation, IRF3 translocated into the nucleus in about 70% of hepatocyte-like cells,

347

but the nuclear translocation of IRF3 was significantly reduced in those HBV-infected

348

cells (using HBsAg as a positive marker for HBV infection)(Fig. 1H). In PHHs which

349

support a higher-efficient viral infection (about 30%-40%) (Fig. 1I, left part), the

350

cGAMP-elicited IFN-β gene production in HBV-infected cells was obviously lower

351

compared with that in uninfected cells (Fig. 1I, right part). The data strongly support

352

the results obtained in the overexpression system that the presence of HBV replication

353

dampened STING-activated signaling pathways.

354

Overall, these results indicated that HBV may have developed some strategies to

355

evade the STING-mediated host antiviral immune responses.

356

2. HBV polymerase (Pol) suppresses STING-induced IFN-β production.

357

To determine which viral proteins are responsible for the inhibition of

358

STING-mediated IFN-β induction by HBV, we performed a functional screening

359

assay by co-transfecting the constructs encoding HBV proteins, including polymerase

360

(Pol), pre-Core, Core, HBx, or HBs together with the STING-expressing plasmid and 12 / 32

361

the reporter plasmids into Huh7 cells. The results showed that only expression of

362

HBV Pol, but not other viral proteins, obviously suppressed the IFN-β promoter

363

activation induced by ectopic expression of STING in Huh7 cells (Fig.2A), and the

364

inhibitory effect was shown in a dose-dependent manner using both of the amino- and

365

carboxy-terminally Flag-tagged Pol constructs (Fig.2B). To further confirm the

366

suppression of Pol on the STING-mediated endogenous IFN-β production, HEK 293

367

cells were co-transfected with plasmids encoding HBV Pol and STING and the

368

cellular mRNA levels of IFN-β and interferon-stimulated gene 56 (ISG56) were

369

quantified by real-time qPCR. The results showed that the expression of Pol resulted

370

in decreased production of both IFN-β and ISG56 mRNAs (Fig.2C). We next

371

examined the effect of HBV polymerase on STING-mediated IRF3 activation in

372

HEK293 cells by detecting

373

hallmark of IRF3 activation and IFN induction (41). The results demonstrated that

374

STING-induced dimerization of IRF3 was inhibited by Pol in a dose-dependent

375

manner (Fig.2D).

the dimerization of IRF3 which is considered as a

376

We further confirmed the inhibitory effect of Pol on the STING-mediated IFN-β

377

induction using cGAMP and poly(dA:dT) as STING activators. First, we used

378

cGAMP as an IFN-β stimulus. Consistently, Pol also inhibited STING-mediated

379

IFN-β promoter activation upon cGAMP stimulation in a dose-dependent manner (Fig.

380

2E). Previous reports have showed that STING plays an important role in the

381

regulation of IFN-β induction in response to transfected poly(dA:dT) which is a

382

synthetic dsDNA that mimics dsDNA virus (21, 42). We verified that

383

poly(dA:dT)-induced IFN-β production is mostly dependent on STING by knocking

384

down STING expression in PH5CH8 cells which has the intact capacity for type I IFN

385

induction (43) (data not shown). Based on this, PH5CH8 cells expressing Pol or not

386

were used to further transfected with poly(dA:dT) to activate the STING signaling.

387

The results showed that less IFN-β mRNA was detected in cells expressing Pol

388

compared to that in control cells upon poly(dA:dT) stimulation (Fig.2F). In addition,

389

the impaired antiviral response mediated by STING was also observed in Huh7 cells

390

expressing Pol (Fig. 2G). 13 / 32

391

To examine whether Pol could act as an IFN- production blocker in the context of

392

HBV replication, we compared the response of Huh7 cells harbouring the wild type

393

pHBV1.3 or the polymerase-null mutant pHBV1.3-∆Pol upon cGAMP stimulation.

394

As shown in Fig.2G, in Huh7 cells transfected with the wild-type HBV, IFN-

395

promoter activation induced by cGAMP was obviously reduced, while not much

396

affected in those cells lacking HBV polymerase expression (Fig.2H). Similar results

397

were obtained in Huh7 cells transfected with either the wild-type pCMV-HBV

398

construct or the corresponding Pol-null mutant pCMV-HBV-∆Pol (data not shown),

399

thereby verifying the inhibitory effect of HBV Pol on the STING-mediated IFN-

400

production in the context of viral replication.

401

Considering Pol has an indispensible role in HBV genome replication as reverse

402

transcriptase, we intended to explore whether the Pol’s catalytic activity is involved in

403

its inhibitory effect on STING signaling. To test this point, we constructed the

404

polymerase-inactive mutant pHBV1.3-YMHD with which none of HBV DNA

405

replication intermediates wasdetected (data not shown) and tested its effect on

406

STING-mediated IFN-β production. We found that the mutated Pol exhibited the

407

comparable suppression effect on the IFN-β promoter activation with the wild type

408

Pol in Huh7 cells (Fig.2I, upper part). A similar observation was obtained with the

409

mutant pFlag-Pol-YMHD (Fig.2I, lower part). Thus, the results indicate that Pol

410

suppresses STING signaling in a manner that is independent of its catalytic activity.

411

Collectively, these results demonstrate that Pol is predominantly responsible for the

412

suppression of STING-mediated IFN- production by HBV in human hepatic cell

413

lines.

414

3. HBV polymerase physically associates with STING.

415

To gain further insight into how HBV polymerase disrupts STING signaling, we

416

determined whether Pol interacts with STING to inhibit its function. We first

417

examined if Pol and STING co-localize in Huh7 cells by immunofluorescence. As

418

shown in Fig. 3A (upper panel), Flag-tagged Pol exhibited a cytoplasmic punctate

419

pattern of immunofluorescence which moderately co-localized with STING, implying

420

that Pol may interact with STING. By contrast, the viral protein Core did not show 14 / 32

421

any co-localization with STING (Fig. 3A, lower panel). In view of these results, we

422

further confirmed the association of Pol with STING by co-immunoprecipitation

423

analysis. The Pol-expressing stable cell line 293T-Flag-Pol and the control cell line

424

293T-GFP were transfected with HA-tagged STING. The results showed that the

425

Flag-tagged Pol, but not the GFP control, could efficiently co-precipitate with the

426

STING protein (Fig. 3B), suggesting that Pol binds to STING. However, we did not

427

detect any interaction between Pol and the retinoic acid inducible gene (RIG-I), which

428

is known as an RNA sensor that can induce IFN-β production and also be inhibited by

429

Pol (10, 11) (Fig. 3C). These results suggested that the association between Pol and

430

STING is specific.

431

4. The RT/RH domain of Pol binds to STING and is responsible for the

432

inhibitory activity of Pol.

433

HBV polymerase is composed of four distinct regions, including the terminal protein

434

(TP), the spacer, the reverse transcriptase (RT), and the ribonuclease H (RNase H). To

435

delineate the region of Pol interacting with STING, a series of truncated mutants of

436

Pol were constructed (Fig. 4A). We co-transfected cells with the plasmids expressing

437

Flag-tagged truncated mutants of Pol together with HA-tagged STING and examined

438

the association between them by co-immunoprecipitation analysis. The results showed

439

that HA-tagged STING could bind to the full-length Pol, RT/RH, RT and RH

440

domains of Pol, but barely to the TP/Spacer region. Moreover, binding of RT/RH and

441

RT domains with STING exhibited high affinity (Fig. 4B, upper panel).

442

To test whether the association of Pol with STING resulted in the inhibition of

443

STING-mediated IFN- production, the truncated mutants of Pol were further

444

co-transfected with the STING-expressing and the IFN-β reporter plasmids into Huh7

445

cells. The results showed that the IFN-β promoter activation was greatly inhibited in

446

the cells expressing full-length Pol, RT/RH, RT and RH but was rarely affected in the

447

cells expressing TP and Spacer regions of Pol. The biggest inhibition of more than 80%

448

was exhibited by the RT/RH region of Pol (Fig. 4C), suggesting that the protein

449

sequence within the RT/RH region of Pol is critical to the inhibitory effect. A similar

450

effect of RT/RH domains on the IRF-regulating p55-C1B promoter activation was 15 / 32

451

shown in Fig. 4D. To further test the inhibitory effect of the RT and RH domains on

452

IFN- production, we conducted the dose response assay using different amounts of

453

the RT and RH mutants. The results demonstrated that just 100ng of the Pol RT

454

domain could decrease the IFN-β promoter activation by almost 70%, meanwhile, the

455

RH mutant also had an inhibitory effect on the IFN- promoter activation albeit at a

456

milder rate (Fig. 4E). Additionally, overexpression of the mutant ∆RH reduced the

457

IFN-β promoter activation by approximately 50% compared to the control, by

458

contrast, ∆RT mutant only modestly downregulated the IFN-β promoter activation

459

(Fig. 4C). Thus, the RT domain of HBV polymerase relatively makes more

460

contribution on the inhibition of STING-mediated IFN-β induction by Pol, while the

461

RH domain is subordinately responsible for the inhibitory effect. These could be

462

combined with the above data to suggest that the binding of Pol to STING associated

463

with its inhibitory activity on interferon induction, and the binding affinity positively

464

correlated with the degree of the inhibitory effect. We thus concluded that Pol may

465

interact with STING through the RT and RH domains and consequently suppresses

466

STING-mediated IFN- induction in the transfection system.

467

5. The K63-linked polyubiquitination of STING is drastically decreased by HBV

468

polymerase.

469

Modification of signaling molecules by ubiquitination plays a central role in

470

transmitting signals important for activation of innate antiviral responses (44). Recent

471

studies indicate that STING polyubiquitination is required for the activation of IFN

472

induction pathway (45, 46). Ubiquitination events structurally contain several distinct

473

ubiquitin chain linkage types (47). The best characterized linkages to date are the K48

474

ubiquitin chain and the K63 ubiquitin chain (48). K48-linked polyubiquitin mainly

475

targets proteins for proteasomal degradation, whereas K63-linked polyubiquitin more

476

often regulates protein function, subcellular localization, or protein-protein interaction.

477

In view of our above observations and to define how the interaction between Pol and

478

STING abrogates the STING-mediated IFN-β production, we first determined if Pol

479

impairs STING polyubiquitination by in vivo ubiquitination analysis in HEK293T

480

cells. A dramatic decrease in STING ubiquitination was observed in cells expressing 16 / 32

481

Pol compared to that in control cells (Fig. 5A). Meanwhile, the expression levels of

482

STING in the presence and absence of HBV Pol expression remained comparable,

483

suggesting that the linkage of STING ubiquitination impaired by Pol is most likely

484

K63 (Fig. 1 B and Fig. 5A, middle panels). To confirm this point, we employed two

485

plasmids including pHA-Ub-K48R, in which the lysine at position 48 of the ubiquitin

486

was substituted with arginine, and pHA-Ub-K63, in which all lysines except K63

487

were mutated to arginines. The cells transfected with HA-Ub-K48R still showed a

488

reduction in the level of ubiquitinated STING in the presence of Pol (Fig. 5B).

489

Moreover, the K63-linked ubiquitination of STING which has been shown to be

490

important for cellular antiviral response (45, 46), was markedly decreased in the cells

491

expressing Pol, indicating that HBV Pol inhibits IFN- production by specifically

492

targeting the K63- rather than the K48-linked ubiquitination of STING (Fig. 5C).

493

Subsequently, we confirmed this in the context of HBV replication using pHBV1.3 or

494

pHBV1.3-∆Pol in Huh7 cells and found that the K63-linked ubiquitination of STING

495

was decreased in the cells transfected with the wild type HBV genome, however, this

496

attenuation was rescued in the cells containing the HBV Pol-null mutant (Fig. 5D).

497

Next, to define which domain(s) of Pol contribute to the disruption of STING

498

ubiquitination, we co-transfected the series of Pol truncated mutants together with

499

STING and HA-Ub-K63 plasmids followed by examination of STING ubiquitination.

500

Expression of truncated mutants of Pol downregulated K63-linked ubiquitination of

501

STING to a different extent, with the biggest decrease exhibited by the full length Pol,

502

RT/RH domain and RT domain, followed by ∆RT and ∆RH mutants and a weak

503

decrease exhibited by TP/Spacer and RH domains (Fig. 5E, top panel). These results

504

were largely in agreement with our previous observations on their inhibitory effects

505

on STING signaling (Fig. 4B-4E) and suggest that the impairment of K63-linked

506

ubiquitination of STING by Pol may depend on the binding of Pol to STING, thereby

507

leading to a weakened IFN-β production and antiviral response.

508

6. HBV polymerase did not affect the expression of the E3ubiquitin ligases

509

TRIM32 and TRIM56 and their interaction with STING.

510

The E3ubiquitin ligases tripartite motif protein 32 (TRIM32) and TRIM56 have been 17 / 32

511

identified to target STING for K63-linked ubiquitination (45, 46). Thus we speculate

512

whether Pol disrupts the K63-linked ubiquitination of STING by competitively

513

binding to STING that associates with these two E3 ligases. To this end, we examined

514

the impact of Pol on the interaction of TRIM32 or TRIM56 with STING by

515

co-immunoprecipitation analysis. The results showed that the expression levels of

516

TRIM32 and TRIM56 were comparable, regardless of the presence or absence of Pol

517

(Fig.6A, input part). Moreover, the interaction between STING and TRIM32 or

518

TRIM56 was not apparently affected by Pol in the overexpression system (Fig. 6A,

519

top panel).To further confirm this result, we pulled down the endogenous TRIM32 or

520

TRIM56 by Flag-tagged STING with or without Pol. Analysis of the

521

immunoprecipitated products showed that Pol had little effect on the binding of

522

STING to endogenous TRIM32 (Fig. 6B). However, we failed to detect the

523

interaction between STING and endogenous TRIM56 in 293 cells, which may be due

524

to the expression of TRIM56 in the cells is rather low (data not shown). Taken

525

together, these data suggested the suppression of STING K63-linked ubiquitination by

526

Pol was not mediated by a disturbance of the expression of TRIM32 or TRIM56 and

527

their interaction with STING.

528 529

Discussion

530

It has long been controversial whether HBV induces innate immune responses during

531

its infection. A previous study using the chimpanzee model showed that HBV didn’t

532

activate the host innate antiviral responses in the liver, and thus it was described as a

533

‘stealth virus’ (49, 50). Nevertheless, several recent observations in clinic specimens,

534

chimeric mouse models and infectious cell models have shown that HBV may induce

535

some type I IFN and type III IFN production, suggesting that the host can sense the

536

HBV infection and then initiates the innate immune responses (51-55). A possible

537

explanation for the conflicts is that HBV has evolved numerous strategies to block the

538

host antiviral responses early in infection (6-11, 56-59) and thus it is hard for the

539

investigators to detect the host innate antiviral responses during its infection. In the

540

present study, we found that HBV could escape host innate immune response via 18 / 32

541

antagonizing IFN-β induction mediated by STING, a key molecule in the host

542

cytosolic DNA-sensing pathways (17, 18, 20, 38, 60). Our results revealed, for the

543

first time, that the HBV polymerase interacts with STING and inhibits its K63-linked

544

ubiquitination associated with loss of STING function and consequent impairment of

545

IRF3 activation and IFN-β induction and antiviral response.

546

Given the key role of STING in the regulation of host antiviral response, many

547

viruses have been reported to target it for subversion of the host innate immunity by

548

various elaborate mechanisms. It has been shown that HCV, also a hepatotropic virus

549

with a high rate of persistence, inhibits STING-mediated signaling through the viral

550

NS4B protein (40, 61). The protease NS2B3 of dengue virus can cleave STING to

551

block the type I IFN induction (62, 63), and the papain-like proteases of coronaviruses

552

interfere with the STING-mediated signaling pathway by acting as viral

553

deubiquitinases (64, 65). For HBV, though the PRRs and related signaling pathways

554

involved in sensing HBV infection and triggering antiviral innate immune responses

555

remain to be clarified clearly, many reports have shown that activation of the PRR

556

pathways specifically inhibit the HBV replication (6, 26, 54, 66, 67). Moreover, DAI,

557

a cytosolic DNA sensor that also utilizes STING as an adaptor (68), was also showed

558

to inhibit HBV replication in our previous work (69), implying that suppression of the

559

activation of STING, the converging point of the PRRs signaling, may be an efficient

560

strategy for HBV, like many other viruses, to evade the innate antiviral activity at an

561

early stage.

562

Although several cellular biological events, such as STING ubiquitination,

563

dimerization and its recruitment of TANK-binding kinase 1 (TBK1) (41, 70), are

564

reported to be critical for STING-mediated signal transduction, more investigations

565

are needed to characterize the detailed mechanisms that control STING activation and

566

signaling (71). In this study, we demonstrated that HBV Pol markedly impaired

567

K63-linked ubiquitination of STING by physically associating with it through the

568

RT/RH region, though had moderate effect on STING dimerization, which is essential

569

for STING self-activation and subsequent downstream signaling culminating in the

570

induction of type I IFN (21) (data not shown). The recruitment of TBK1 and IRF3 by 19 / 32

571

STING is also required for the activation of STING signaling and appears to be a

572

process that regulated by STING ubiquitin modification. Although K63-linked

573

ubiquitination of STING was significantly inhibited by Pol, the results obtained in

574

293T cells didn’t show that Pol could affect the interaction between overexpressed

575

TBK1 and STING (data not shown). Since the K63-ubiquitination of STING is not

576

only important for STING to recruit TBK1/IRF3, but also critical for STING as a

577

scaffold protein to promote TBK1 and IRF3 activation, the observations we’ve found

578

that Pol-mediated disruption of K63-linked ubiquitination of STING didn’t affect the

579

recruitment of TBK1 to STING but resulted in the significant inhibition of IRF3

580

phosphorylation (data not shown) and dimerization and IFN induction were

581

reasonable. Additionally, while the essential role of STING in DNA sensing pathways

582

is well-established, it has also been shown to play a role in RIG-I-mediated IFN-β

583

production (22, 24). Furthermore, HBV Pol has been reported to inhibit

584

RIG-I-mediated signaling by disrupting the interaction between TBK1/IKKε and

585

DDX3 (10, 11). Therefore, there is a possibility that the suppression effect of Pol on

586

RIG-I signaling is involved in its antagonism on STING-mediated IFN-β induction.

587

To test this hypothesis, we examined whether overexpression of DDX3 could restore

588

the inhibitory effect of HBV polymerase on STING signaling and found that the

589

suppressed IFN-β promoter activity could be efficiently rescued by over-expressed

590

DDX3 when RIG-I but not STING acted as the stimulator (data not shown). This

591

result partly exclude the possibility that Pol inhibits STING signaling to a large extent

592

through its act on RIG-I signaling. Therefore, we concluded that HBV Pol inhibits

593

STING-mediated type I IFN production mainly by dampening its K63-linked

594

ubiquitination. Nevertheless, the precise delineation how Pol affects STING-mediated

595

signaling requires further understanding of the molecular mechanisms involved in

596

STING activation.

597

HBV Pol consists of four domains. The RT domain is responsible for reverse

598

transcription and nucleocapsid assembly of HBV (72-74), while the TP domain serves

599

as a protein primer to initiate reverse transcription (75). The C-terminal RNase H

600

domain directly mediates the degradation of pregenomic RNA template after reverse 20 / 32

601

transcription to maintain the stability of HBV genome. It should be noted that Pol is

602

commonly believed to be inefficiently translated and rapidly encapsidated. However,

603

some investigators detected non-encapsidated polymerase accumulated in the

604

cytoplasm in a manner similar to non-encapsidated duck hepatitis B virus (DHBV)

605

polymerase (76, 77), which implies that Pol may have additional functions besides

606

reverse transcriptase. Recent reports implicate Pol displays immunomodulatory

607

activities through binding to a series of host factors (78). Besides blocking RIG-I- and

608

TLR3-mediated IFN-β induction via dampening the interaction between TBK1/IKKε

609

and DDX3 as mentioned above, Pol was also shown to abrogate IFN-α signaling by

610

suppressing the nuclear translocation of STAT1 and STAT2 in Huh7 cells (79).

611

Interestingly, the detailed studies of the TP domain of Pol revealed its association with

612

PKC-δ resulting in inhibition of PKC-δ phosphorylation, meanwhile, RH domain was

613

able to interfere with nuclear transportation of STAT1/2 through a competitive binding

614

to importin-α5 (80). Here, we demonstrated that the RT and RH domains of Pol were

615

responsible for the inhibition of STING-activated signaling transduction through

616

associating with STING and inhibiting its activation. Overall, these investigations

617

support a notion that besides its inherent catalytic role in viral replication, Pol has

618

multifunctional activities, including regulating host immune signaling pathways by

619

binding to diverse host targets, which may contribute to HBV evasion of the innate

620

immunity. Nevertheless, further work should aim to determine the significance of

621

Pol-mediated immune modulation during HBV infection at more physiological levels

622

of Pol expression in vivo.

623

In summary, a new role of HBV polymerase in inhibition of IFN-β production

624

through selectively targeting the cytosolic DNA sensor and the adaptor molecule

625

STING has been proposed here. We believe that these findings may promote our

626

understanding on the molecular mechanisms by which HBV escapes the innate

627

immunity and establishes chronic infection and provide new target for designing

628

novel therapeutic interventions to combat HBV.

629 630

Acknowledgments 21 / 32

631

We thank Dr. Takashi Fujita and Dr. Hiroki Kato (Institute for Virus Research, Kyoto

632

University, Kyoto, Japan) for the insightful suggestions and kindly providing the

633

plasmids (pEF-STING, pEF-STING-Myc, and p-55C1B-Luc). We also thank Dr.

634

Hongbing Shu (The College of Life Sciences, Wuhan University, Wuhan, China) for

635

his generous gift of the plasmids (pHA-MITA, pFlag-MITA, pFlag-TRIM32,

636

pFlag-TRIM56 and the RNAi plasmids targeting human STING) and the

637

anti-TRIM32 antibody. We are indebted to Dr. Shiyan Yu for good advice and full

638

support. We are grateful to Dr. Zekun Wang for preparing several of the truncations

639

of Pol and the ubiquitin mutants pHA-Ub-K48R and pHA-Ub-K63 used in this study

640

and helpful discussions. We thank Dr. Maya Kozlowski for critical review of the

641

manuscript.

642

This research was supported by the National Key Basic Research Program of China

643

(2012CB519000 to Z.Y.), the National Megaprojects of China for infectious Diseases

644

(2012ZX10002007-001 to Z.Y.), the German Research Foundation (SFB/Transregio

645

TRR60 to Z.Y.), the National Natural Science Foundation of China (31200129 to J.

646

Li), the China Postdoctoral Science Foundation (201104232 to J. Li), and the China

647

Postdoctoral Science Foundation (2014M551325 to J.L. Chen).

648 649

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894

Figure legends

895

Fig.1 HBV inhibits STING-mediated IFN-β induction and antiviral response.

896

(A, B) Huh7 cells were co-transfected with pIFN-β-Luc (A) or p-55C1B-Luc (B),

897

pRL-TK and 100ng, 200ng, 400ng of the plasmids pHBV1.3 or pCMV-HBV together

898

with pSTING (80ng) or empty vector. After 36h, the cells were extracted and assayed

899

for luciferase activity. The expression of STING in cells was examined by Western

900

blot and the expression of β-actin was examined as a loading control. Meanwhile, the

901

level of core capsids was detected using anti-Core antibody as described in Materials

902

and Methods (A, right part). (C) Measurement of endogeous STING expression in the

903

indicated cell lines by Western blot with anti-STING antibodies. (D) Huh7 cells were

904

co-transfected with pIFN-β-Luc, pRL-TK, and 125ng, 250ng, 500ng of the plasmids

905

pHBV1.3 or pCMV-HBV together with pSTING (25ng) or empty vector. After 36h,

906

the cells were mock-treated or treated with the indicated concentration of cGAMP for

907

additional 16h and then harvested for luciferase assay. Data are expressed as the mean

908

fold induction ± SD relative to control levels. (E, F) Huh7 cells were co-transfected

909

with 125ng, 250ng, 500ng of pHBV1.3 together with pSTING (25ng) or empty vector.

910

After 36h, the cells were mock-treated or treated with 100nM cGAMP for 16h. The

911

amount of IFN-β in the supernatants was determined by ELISA (E). Meanwhile, the

912

remaining supernatants from Huh7 cells were then collected and transferred to Vero

913

cells for overnight incubation with a positive control using IFN-a (500 IU ml-1)

914

treatment. Vero cells were then infected with NDV-GFP (80 HAU ml-1) for additional

915

24h. The GFP expression levels were determined by microscopy observation and

916

Western blot (F). (G) HepAD38 cells cultured in the presence or absence of

917

doxcycline were co-transfected with pIFN-β-Luc, pRL-TK and pSTING (25ng) or 28 / 32

918

empty vector. After 36h, the cells were mock-treated or treated with the indicated

919

concentration of cGAMP for additional 16h and then harvested for luciferase assay.

920

(H) HepaRG cells were seeded onto the chambered coverglass and proliferated and

921

differentiated. At day 10 post HBV infection of the dHepaRG cells, the cells were

922

mock-treated or treated with 200nM cGAMP for an IRF3 nuclear translocation assay

923

by immunofluorescence (left part). The number of cells with or without nuclear IRF-3

924

from HBs (-) and HBs (+) hepatocyte-like cells in three different visual fields at low

925

magnification was counted and presented as the mean percentage + SD (right part). (I)

926

PHHs were seeded onto the chambered coverglass or 24-well plates. At day 10 post

927

HBV infection, the cells were either collected for the immunofluorescence (left part)

928

or mock-treated or treated with 200nM of cGAMP for 6h and then harvested for the

929

measurement of IFN-β mRNA by quantitative RT-PCR (right part). The results are

930

representative of three independent experiments.* P