JVI Accepted Manuscript Posted Online 4 February 2015 J. Virol. doi:10.1128/JVI.03094-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Inhibition of Hepatitis B Virus Gene Expression and Replication by Hepatocyte

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Nuclear Factor 6

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Running Title: HNF6 inhibits HBV replication

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Ruidong Hao1, Jing He1, Xing Liu1, Guozhen Gao3, Dan Liu2, Lei Cui1, Guojun Yu1,

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Wenhui Yu1, Yu Chen1, Deyin Guo1, 2,#

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1

State Key Laboratory of Virology and Modern Virology Research Center, College of

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Life Sciences, Wuhan University, Wuhan, Hubei 430072, China

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2

School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430072, China

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3

Department of Molecular Carcinogenesis, the University of Texas MD Anderson Cancer

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Center, Smithville, TX 77030, USA

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#

Address correspondence to Deyin Guo, [email protected]

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Abstract word count: 198 words

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Text word count: 4670 words

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Abstract

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Hepatitis B virus (HBV), a small enveloped DNA virus, chronically infects more than

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350 million people worldwide and causes liver diseases from hepatitis to cirrhosis and

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liver cancer. Here, we report that hepatocyte nuclear factor 6 (HNF6), a liver-enriched

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transcription factor, can inhibit HBV gene expression and DNA replication.

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Overexpression of HNF6 inhibited, while knockdown of HNF6 expression enhanced

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HBV gene expression and replication in hepatoma cells. Mechanistically, SP2 promoter

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was inhibited by HNF6, which partly accounts for the inhibition on S mRNA. Detailed

29

analysis showed that a cis element on HBV genome (nt 3009-3019) was responsible for

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the inhibition of SP2 promoter by HNF6. Moreover, further analysis showed that HNF6

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reduced viral pregenomic RNA (pgRNA) posttranscriptionally via accelerating the

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degradation of HBV pgRNA independent of La protein. Furthermore, by using truncated

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mutation experiments, we demonstrated that the N-terminal region of HNF6 was

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responsible for its inhibitory effects. Importantly, introduction of an HNF6 expression

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construct with HBV genome into the mouse liver using hydrodynamic injection resulted

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in a significant reduction in viral gene expression and DNA replication. Overall, our data

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demonstrated that HNF6 is a novel host factor that can restrict HBV replication via both

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transcriptional and posttranscriptional mechanisms.

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Importance

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HBV is a major human pathogen whose replication is regulated by host factors. Liver

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enriched transcription factors are critical for many liver functions including metabolism,

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development and cell proliferation, and some of them have been shown to regulate HBV

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gene expression or replication in different manners. In this paper, we showed that HNF6

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could inhibit the gene expression and DNA replication of HBV via both transcriptional

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and posttranscriptional mechanisms. As HNF6 is differentially expressed in men and

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women, the current results may suggest a role of HNF6 in the gender dimorphism of

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HBV infection.

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Introduction

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Hepatitis B virus (HBV) is a noncytopathic, hepatotropic virus that targets the liver and

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replicates in hepatocytes (1). Around 2 billion people have been infected with HBV

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worldwide, and 350 million people are chronic carriers suffering from a higher risk of

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chronic liver diseases, including hepatitis, cirrhosis and hepatocellular carcinoma (2).

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Upon infection of hepatocytes, the viral relaxed circular DNA (rcDNA) genome is

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delivered into the nucleus and repaired to form covalently closed circular DNA (cccDNA).

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The cccDNA serves as the transcription template of pregenomic RNA (3.5 kb), and other

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mRNAs including precore RNA (3.5 kb), S RNAs (2.4/2.1 kb), and X RNA (0.7 kb). The

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transcripts are exported to cytoplasm and translated into viral proteins. After encapsidated

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by core and polymerase protein, the pregenomic RNA is reverse transcribed to viral DNA

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by the viral polymerase. The newly formed relaxed circular DNA is either recycled to the

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nucleus to amplify the cccDNA pool or enveloped by viral surface proteins and secreted

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as virion (reviewed in ref (3-5) ).

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Liver enriched transcription factors (LETFs) control transcription of liver specific genes,

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which are involved in multiple physiological processes including metabolisms,

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differentiation, and development (6, 7). According to their functional domains, the LETFs

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are grouped into several families including hepatocyte nuclear factor 1 (HNF1), forkhead

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box A2 (FoxA2, previously called HNF3β), HNF4, HNF6, CCAAT/element binding

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proteins (C/EBPs), and D site binding protein (6, 7). Apart from their physiological roles

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in cells, many LETFs, including HNF1, FoxA2, HNF4, and C/EBPs, have been shown to

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regulate HBV replication transcriptionally or posttranscriptionally (8-10).

73 74

HNF6, the prototype of the one-CUT transcription factor family, is characterized by a

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divergent Homeo domain and a single CUT domain at the C terminus (11, 12). The

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expression of HNF6 is high in the liver and low in the spleen, brain, and testis (12).

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HNF6 controls the expression of many liver-specific genes, which are involved in the

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glucose metabolism, bile homeostasis, and hepatic cell proliferation (13). The expression

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of HNF6 is regulated by growth hormone (GH) and the GH-HNF6 pathway has been

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shown to be imperative for liver protection against chronic injury through regulating the

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expression of genes involved in proliferation (14-16). The GH-HNF6 pathway also

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contributes to the gender disparity via regulating the expression of some

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female-predominant genes (17). The molecular mechanisms involved in gene regulation

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by HNF6 are either by directly binding to the promoter region of its target genes or by

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cooperating with other proteins (13).

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Although many LETFs have been shown to participate in HBV replication, whether

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HNF6 influences HBV replication is unknown. Herein, we provide evidences that ectopic

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expression of HNF6 significantly inhibits HBV gene expression and replication in vitro

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and in vivo. It was shown that HNF6-mediated HBV inhibition occurred both at

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transcriptional and posttranscriptional level through inhibiting SP2 promoter-mediated

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transcription and accelerating the decay rate of pgRNA.

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Materials and methods

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Plasmids.

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The HBV (genotype D, Genbank accession number V01460.1) replication-competent

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plasmid, pHBV1.3, was a generous gift from Dr. Guangxia Gao (Institute of Biophysics,

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Chinese Academy of Sciences, China). Plasmids pSV-pgRNA, pSV2.4, pSV2.1 and

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pCMV-pgRNA, pCMV-2.4, pCMV-2.1 containing indicated fragments HBV genome

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were constructed as previously reported (referred to Fig. 6A for schematic illustration)

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(18, 19). pTRE2-HBV was generated by insertion of HBV pgRNA fragment

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(1812-3182/1-1997) into the vector pTRE2 (Clonetech) between NheI and HindIII. To

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construct the reporter plasmids, the HBV DNA fragments EnhI/X (nt 950-1375), EnhII/C

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(nt 1415-1815), SP1 (2707-2849), SP2 (2937-3182) and its serial deletion mutants

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(referred to Fig. 5A) were PCR amplified from pHBV1.3 and inserted into the SacI and

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HindIII restriction sites of pGL3-Basic (Promega, Madison, WI). The pHBV1.3/S-Luc

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was constructed based on the pHBV1.3 backbone, in which the S open reading frame

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(ORF) was replaced with firefly luciferase ORF with the unique restriction sites XhoI and

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NsiI on pHBV1.3. The N-terminal HA-tagged HNF6 fragment was PCR amplified from

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HepG2 cDNA and inserted in the pcDNA3.1 Vector (Invitrogen, Carlsbad, CA). The

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deletion mutants of HNF6 were constructed by PCR methods. Two HNF6 short hairpin

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RNAs (shRNA) and control shRNA targeting enhanced green fluorescence protein

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(shGFP) were inserted into vector pLKO.1. The targets of indicated shRNAs are

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5’-AAGACTTAGCTCACCTGCATT-3’ (shHNF6-1), 5’-AACCCTGGAGCAAACTCAAAT-3’

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(shHNF6-2) and 5’-GGCGATGCCACCTACGGCAAG-3’ (shGFP).

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Cell cultures and Transfection.

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Human hepatoma cell-derived HepG2 and Huh7 and human embryonic kidney 293T cells

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were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal

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bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. HepG2 and Huh7 cells

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were transfected with Neofect (Neofect Biotech, Beijing, China) according to the

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manufacturer’s instructions. For producing HNF6 shRNAs, HEK 293T cells were

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cotransfected with shRNA expressing plasmids separately with packaging plasmids

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pMD2.G and psPAX using the calcium phosphate precipitation method. And the

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lentiviruses were harvested 48 h and 72 h post transfection.

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HBV DNA analysis.

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HepG2 cells were cotransfected with pHBV1.3, pSV-β-gal and HNF6 or its control vector

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pcDNA3.1 in the amounts as indicated in the related figures or legends. 96 hours post

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transfection, the cells were washed with cold PBS, and lysed in NP40 lysis buffer (50

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mM Tris-Hcl, pH 7.0, and 0.5% NP40) at 4°C. The same volume of each lysate was

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subjected to β-gal activity assay and served as transfection efficiency control. Followed

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by centrifugation, the supernatants were digested with DNaseI (Promega, Madison, WI)

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at 37°C for 1 hour and then the enzymes were inactivated at 70°C for 15 minutes in the

135

presence of 10 mM EDTA. Then the core-associated DNA were isolated by proteinase K

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digestion, phenol-chloroform extraction, ethanol precipitation, and resolved in 20 μl TE

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buffer. All of extracted DNA were loaded and separated in a 0.8% agarose gel. After

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standard denaturation and neutralization procedures, the DNA was transferred onto a

139

positively charged nylon membrane (GE Healthcare, Waukesha, WI) and probed with a

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DIG-labeled HBV RNA probe. The probe preparation and subsequent DIG detection

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were conducted with DIG Northern Starter Kit (Roche Diagnostics, Indianapolis, IN)

142

according to the manufacturer’s instructions.

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Extracellular encapsidated HBV DNA was extracted according to the protocol described

144

previously (20) with modifications. Briefly, 10 μl culture supernatants or mice sera were

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treated with DNaseI for 1 hour at 37°C to remove free DNA and plasmids. After the

146

enzyme was inactivated at 75°C for 15 min in the presence of EDTA, the samples were

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147

added into 100 μl lysis buffer (20 mM Tris-Hcl, 20 mM EDTA, 50 mM NaCl, and 0.5%

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SDS) containing 27 μg proteinase K. After incubation at 65°C overnight, viral DNA was

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isolated by phenol/chloroform extraction and ethanol precipitation. The DNA pellet was

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rinsed with 70% ethanol and resuspended in 10 μl TE. Then the viral DNA was analyzed

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by qPCR.

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Western Blotting.

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The transfected cells were lysed in RIPA buffer with sodium dodecyl sulfate (SDS). Then

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the protein samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE)

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and transferred onto a nitrocellulose membrane (GE Healthcare, Waukesha, WI). The

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membrane were then blocked with skim milk and probed with indicated antibodies. The

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immunoblot signals were detected by enhanced chemiluminescence (ECL) substrate

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(Millipore, Billerica, MA). Antibodies used in this study include anti-HA (Sigma,

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St. Louis, US), anti-HNF6 (Proteintech, Wuhan, China), anti-La (Proteintech, Wuhan,

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China), and anti-β-actin (Proteintech, Wuhan, China).

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Real-time PCR.

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For mRNA detection, total RNA was isolated from cells or liver tissues using TRIzol

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reagent. The RNA was reverse transcribed to first strand cDNA using M-MLV reverse

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transcriptase (Promega, Madison, WI). The SYBR Green master mix (Roche Diagnostics,

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Indianapolis, IN) was used for real-time PCR. GAPDH mRNA was analyzed to serve as

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internal

control.

The

primers

used

in

this

study

were:

2RC/CCS

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5’-CTCGTGGTGGACTTCTCTC-3’ and 2RC/CCAS 5’-CTGCAGGATGAAGAGGAA-3’ for

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HBV-DNA amplification (21); 5’-ACCGCCAGTCAGGCTTTCTTTC-3’ and 5’-TCACTTAC

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GCCAATGTCGTTATCC-3’ for β-gal mRNA detection; 5’-CCACTCCTCCACCTTTGAC-3’

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and 5’-ACCCTGTTGCTGTAGCCA-3’ for GAPDH detection.

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Cytoplasmic and Nuclear RNA isolation.

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Cell fraction was conducted according to the protocol in ref (22). Briefly, 48 hours post

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transfection, cells were harvested and lysed in lysis buffer (50 mM Tris-Hcl, pH8.0, 0.5%

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NP40, 100 mM NaCl, 5 mM MgCl2, 1 U/μl RNAsin, 1 mM DTT) at 4°C for 5 min. Then

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the nuclei were precipitated by centrifuge at 12000 rpm for 2 min. Cytoplasmic RNA in

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the supernatants was extracted using RNA clean kit (Tiangen Biotech, Beijing, China).

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The nuclear RNA was extracted from nuclei pellets using TRIZol reagent (Invitrogen,

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Carlsbad, CA) according to the manufacturer’s instruction. Then the nucleus and

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cytoplasmic RNA were subjected to Northern blot analysis.

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Analysis of secreted HBV antigens.

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At the indicated time points, cell culture supernatants or mice sera were collected to

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detect the levels of hepatitis B surface antigen (HBsAg) and hepatitis B E antigen

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(HBeAg) by commercial ELISA kit (Kehua Bio-engineering, Shanghai, China). All the

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values were normalized against the β-galactosidase activities in the cell lysates measured

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by Beta-Glo kit (Promega, Madison, WI).

190 191

Immunohistochemistry.

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Paraffin-embedded liver sections were treated with 3% hydrogen peroxide and blocked

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with 5% BSA. The sections were then incubated sequentially with anti-HBc antibody

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(Sigma, St. Louis, US), biotin-labeled secondary antibody, and avidin-biotin complex.

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Peroxidase stain was developed with 3,3’-diaminobenzidine solution and counterstained

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with hematoxylin.

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Dual-Luciferase assays.

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HepG2 cells were cotransfected with HBV reporters (200 ng) together with indicated

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amounts of HNF6 expression plasmids or its control vector, and pRL-TK (50 ng) were

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included as transfection efficiency control. 48 hours post transfection, the cells were lysed

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and subjected to luciferase activity assays using the dual-glo system (Promega, Madison,

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WI).

204 205

Northern blot analysis.

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Total cellular RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA)

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according to the manufacturer’s instructions. 5 µg of total RNA was resolved in 1%

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agarose gel containing 2.2 M formaldehyde and transferred onto positively charged nylon

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membrane (GE Healthcare, Waukesha, WI). To detect HBV transcripts, the membrane

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210

was probed with a DIG-labeled plus strand specific RNA probe corresponding to the

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nucleotides from 156 to 1061 of HBV genome. The probe preparation and subsequent

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DIG detection were conducted with DIG Northern Starter Kit (Roche Diagnostics,

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Indianapolis, IN) according to the manufacturer’s instruction. The amounts of 28S and

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18S rRNA were used as loading controls.

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Hydrodynamics-Based Transfection in Mice.

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For the in vivo experiments, 5-week-old male Balb/c mice were used and separated to 2

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groups (8 each). The pHBV1.3 (10 μg) and pSV-β-gal (5 μg) were injected into tail vein

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of mice together with HNF6 expression construct or empty vector pcDNA3 (20 μg)

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within 8 seconds in a volume of saline equivalent to 10% of mouse body weight. Animals

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were sacrificed after 4 days. Sera were taken for analysis of HBsAg, HBeAg, HBV-DNA,

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and alanine aminotransferase (ALT). For Northern blot analysis, a piece of liver tissue

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was homogenized in 1 ml of TRIZol reagent and total RNA was isolated following the

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manufacturer’s protocol. Ten μg of total RNA of each mouse was subjected to Northern

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blot analysis as described above. The level of β-gal mRNA in the liver was determined by

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real-time PCR and served as control for the injection efficiencies. All mice were housed

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in a pathogen-free mouse colony and the animal experiments were performed according

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to the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, P.

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R. China, 1998).

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Results

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Overexpression of HNF6 inhibits HBV gene expression and replication in hepatoma

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cell lines

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To investigate the role of HNF6 in HBV gene expression, a hepatoma cell line HepG2

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was cotransfected with a HBV replication competent plasmid (pHBV1.3) and different

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doses of HNF6 expression vector (pcDNA-HA-HNF6). The expression levels of HNF6

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were detected by Western blot (Fig. 1A). The HBV transcripts in the transfected cells

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were analyzed by Northern blot. As shown in Fig. 1B, the steady levels of HBV RNA

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were remarkably reduced upon HNF6 overexpression. The 3.5 kb transcripts consisting

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of the pgRNA and the longer precore RNA were inhibited in a dose-dependent manner

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(Fig. 1B). Moreover, the 2.4 and 2.1 kb RNAs were inhibited more potently (Fig. 1B).

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Consistent with the reduction of HBV RNAs, the secreted viral protein levels in the

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extracellular culture medium were reduced as well (Fig. 1D and E). In accordance with

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the more potent effects on S mRNA, the inhibitory effect of HNF6 on the production of

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HBsAg was more potent than that of HBeAg. The modest effects on HBeAg might be

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attributed to the differential regulation of precore RNA and pgRNA, as shown previously

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for another LETF, HNF3β (9, 23).

248 249

To further investigate the effect of HNF6 on HBV replication, the replication intermediate

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DNA in the transfected cells and secreted HBV DNA in the culture supernatants were

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isolated and detected by qPCR. As shown in Fig. 1C and 1E, both the cellular and

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extracellular core-associated HBV DNA were reduced upon HNF6 overexpression. Taken

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together, these results demonstrated that overexpression of HNF6 inhibits the gene

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expression and replication of HBV.

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To determine whether the suppressive effect of HNF6 was limited to hepatocyte-derived

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cell lines, pHBV1.3 and HNF6 plasmids were cotransfected into other hepatic or

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non-hepatic cell lines. Results showed that the levels of HBV RNA were inhibited by

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HNF6 overexpression in another hepatoma cell line Huh7 (Fig. 1G), but not in

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non-hepatic cell line HEK 293T (Fig. 1H). These results suggested that HNF6 might

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inhibit HBV replication through hepatocyte-specific genes or pathways.

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As the activity of HNF6 may be affected by the expression level of HNF6, we measured a

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more detailed dose effect of HNF6 on HBV biosynthesis. As shown in Fig. 2,

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cotransfection of 10 ng or 40 ng HNF6 expression plasmid (the HBV DNA:HNF6 ratios

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are 40:1 and 10:1, respectively) were able to reduce HBV RNA (about 3 folds) and

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replication intermediates (about 5 folds). And higher amounts of HNF6 expression

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plasmid exerted a more profound inhibition effects. The expression level of HNF6 in the

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transfected cells have been determined by western blot with an HNF6 antibody (Fig. 2C).

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These results indicate that HNF6 can inhibit HBV replication in a dose-dependent

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manner.

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Knock-down of endogenous HNF6 leads to increased HBV replication

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To further illustrate the role of endogenous HNF6 on HBV, the HNF6 expression in

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HepG2 cells was depleted by transducing with lentiviral vector-based short hairpin RNAs

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(shRNA). As shown in Fig. 3A, the protein levels of HNF6 were markedly reduced in

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cells transduced with shHNF6-1 or shHNF6-2 compared with the control. And the HNF6

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depletion was associated with increased expression of HBV RNAs and replication

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intermediate DNA (Fig. 3B and 3C). Accordingly, the secreted HBsAg and HBeAg levels

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in the culture medium of shHNF6-1 and shHNF6-2 were also elevated significantly (Fig.

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3D and 3E). Overall, these results indicated that endogenous HNF6 possesses the ability

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to restrict HBV gene expression.

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HNF6 suppresses the activity of preS2/S promoter

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To investigate the mechanisms involved in HNF6-mediated HBV suppression, the effects

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of HNF6 on four HBV promoters were determined by a luciferase-based reporter assay.

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HepG2 cells were cotransfected with HNF6 expression plasmid and the reporters driven

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by EnhI/Xp, EnhII/Cp, SP1 and SP2 promoters respectively. As shown in Fig. 4A, HNF6

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overexpression did not affect the activities of HBV enhancerI/X, enhancerII/C, or SP1

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promoters (Fig. 4A). However the activity of SP2 promoter was inhibited by HNF6 in a

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dose-dependent manner (Fig. 4A and B). To further validate its inhibitory effect on S

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promoter, another reporter, pHBV1.3/S-Luc, was constructed based on pHBV1.3, in

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which the major S ORF was substituted by firefly luciferase ORF. Consistent with the

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inhibitory effect above, HNF6 suppressed the activity of pHBV1.3/S-Luc in a

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dose-dependent manner (Fig. 4C). These results indicated that HNF6 could suppress

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HBsAg expression transcriptionally.

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In order to further investigate the mechanisms of the inhibitory effects of HNF6 on the

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HBV SP2 promoter, a serial of deletion mutants of the SP2 were constructed (Fig. 5A).

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As shown in Fig. 5B, deletion of region 3008-3019 rendered the SP2 promoter resistance

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to the inhibition by HNF6. Thus, the region from 3008 to 3019 of HBV genome might be

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responsible for the inhibitory effects of HNF6. However, computer based bioinformatic

303

analysis did not reveal any HNF6-binding site in the SP2 promoter and the flanking

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region of 200 bp. Gel shift experiments also confirmed that HNF6 did not bind the SP2

305

promoter directly (Fig. 5C and 5D). These results suggested that HNF6 does not affect

306

the activity of SP2 directly and it may function through some undefined factor/factors

307

involving a cis element of HBV genome (nt 3008-3019).

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HNF6 reduces the steady state levels of HBV RNA via posttranscriptional mechanisms

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The pgRNA of HBV serves as the template for reverse transcription and is critical for

311

viral replication. The transcription of pgRNA is controlled by the EnhI/X and EnhII/C

312

promoters. Since HNF6 does not influence the activity of these two elements (Fig. 4A),

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we sought to clarify whether the intrinsic promoters of HBV are required for the

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inhibition by HNF6. To this end, we generated three constructs (pSV-HBV, pSV-2.4 and

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315

pSV-2.1), in which the pgRNA, 2.4kb, and 2.1 kb RNAs of HBV are transcribed under

316

control of SV40 promoter as depicted in Fig. 6A. These three constructs were transfected

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into HepG2 cells respectively along with HNF6 or its control vector. As shown in Fig. 6B,

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the levels of HBV RNA transcribed from SV40 promoters were also inhibited by HNF6

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dramatically. To exclude the possibility that HNF6 might affect the activity of SV40

320

promoter directly, a construct in which the β-gal ORF is under control of SV40 promoter

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was transfected into cells along with HNF6 or its control vector. Interestingly, HNF6 did

322

not inhibit the activities of β-gal, indicating that HNF6 does not influence the activity of

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SV40 promoter (Fig. 6D). Similar results were obtained when the SV40 promoter was

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replaced with the CMV-IE promoter (Fig. 6C and 6E). These results suggest that HNF6

325

suppresses the level of 3.5 and 2.4 kb RNAs via posttranscriptional mechanisms. Since

326

the SP2 promoter was also inhibited (Fig. 4D), it is suggested that the 2.1 kb RNA might

327

be suppressed through both transcriptional and posttranscriptional mechanisms.

328 329

HNF6 accelerates the decay rates of HBV RNA independent of La

330

As the HNF6-mediated HBV RNA reduction occurs post-transcriptionally, we suggest

331

that HNF6 may promote the turnover rate of HBV pgRNA. To this end, we directly

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measured the decay rates of HBV pgRNA in the absence or presence of HNF6

333

overexpression. HepG2 cells were cotransfected with HNF6 and pTRE2-HBV, in which

334

the HBV pgRNA transcribed from a tetracycline controlled promoter. Doxycycline was

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added to culture supernatants 36 h after transfection to turn off the HBV pgRNA

17

336

transcription. Then the cells were harvested 0, 3, 6, and 9 hours after the doxycycline

337

addition and subjected to RNA extraction and Northern blot analysis. As shown in Figure

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7A and 7B, the half-life of HBV pgRNA was shortened by HNF6 by about 3 hours in

339

comparison with that of cells transfected with the empty control plasmid (pcDNA3). This

340

result demonstrated that HNF6 could promote the decay rates of HBV pgRNA.

341 342

It is well characterized that upon treatment with cytotoxic T-lymphocyte and some

343

cytokines, the La protein became fragmented and left the HBV RNA vulnerable to

344

endonuclease degradation (24, 25). Then we sought to determine whether the

345

HNF6-mediated pgRNA decay is mediated by La. Our results showed that overexpression

346

of HNF6 did not affect the expression or fragmentation status of La (Fig. 7C). To further

347

verify

348

La-binding-deficient HBV pgRNA, 2.4 and 2.1 kb RNAs constructs were constructed as

349

depicted previously (25, 26). However, HNF6 was also able to inhibit the levels of

350

La-binding-deficient HBV pgRNA, 2.4 and 2.1 kb RNAs (Fig. 7D). These results

351

suggested that the HNF6 mediated HBV RNA degradation is independent of the La

352

protein.

the

effects

of

La

during

HNF6-mediated

pgRNA

degradation,

the

353 354

HNF6-mediated HBV RNA inhibition occurs primarily in the nucleus

355

To further determine the cellular localization where the inhibitory effect occurs, nuclear

356

and cytoplasmic RNA were fractioned and extracted from cells cotransfected with

18

357

pHBV1.3 and HNF6. As shown in Fig. 7E and 7F, the levels of HBV transcripts were

358

reduced in both the nuclear and cytoplasmic portions with similar inhibition rates. Since

359

HBV RNA is transcribed in the nucleus and then exported to the cytoplasm, it is

360

suggested that HNF6-mediated HBV RNA inhibition is primarily a nuclear event.

361 362

The N-terminal domain of HNF6 is required for the anti-HBV activity of HNF6

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HNF6 contains the CUT and Homeo domains at the C-terminal half, which are both

364

involved in DNA binding and protein interactions. The N-terminal part of HNF6 has been

365

shown to interact with cellular factors (27). To further delineate the mechanism involved

366

in the inhibitory effects of HNF6, the contribution of different domains of HNF6 were

367

determined. As shown in Fig. 8A and 8B, the CUT-homeodomain of HNF6 alone was not

368

able to reduce HBV transcripts. While the deletion of the Homeo, CUT domain or both

369

could suppress the levels of HBV transcripts. The immunofluorescence results showed

370

that the N-terminal part of HNF6 is localized in both nucleus and cytoplasm, and the wild

371

type HNF6 is localized exclusively in nucleus (Fig. 8C). In accordance with the northern

372

blot results, the N-terminal domain of HNF6 was also able to inhibit the activity of the

373

SP2 promoter (Fig. 8D) and reduce the amount of HBV nuclear mRNA (Fig. 8E). These

374

results suggested that the N terminal domain accounts for the anti-HBV activity of HNF6.

375 376

HNF6 inhibits HBV gene expression and replication in mice

377

Because HNF6 was shown to inhibit HBV replication in hepatoma cells, we further

19

378

investigate the effects of HNF6 expression on HBV replication in mouse model.

379

pHBV1.3 and HNF6 expression plasmid or its control vector were co-delivered into mice

380

through hydrodynamic injection and the mice were sacrificed 4 days post the injection.

381

The expression of HNF6 in mice livers were determined by Western blot (Fig. 9E).

382

Consistent with the results in the hepatoma cell lines, the titers of HBsAg, HBeAg, and

383

HBV-DNA in mice sera were reduced significantly upon HNF6 injection (Fig. 9A and B).

384

Northern blot result showed that the HBV transcripts were reduced significantly upon

385

HNF6 overexpression (Fig. 9C). Immunohistochemical staining showed that HNF6 could

386

inhibit the HBcAg expression in mice livers (Fig. 9D). Taken together, these results

387

demonstrated that HNF6 is able to inhibit HBV gene expression and replication in vivo.

388 389

Discussion

390

LETFs are important transcription factors involved in liver development, differentiation,

391

and metabolisms (6, 7). Many of them have been shown to regulate HBV gene expression

392

or replication. HNF1α activates the transcription from EnhII/core promoter and thus

393

promotes HBV replication (8). HNF4α activates transcription from EnhII/C, SP1 and SP2

394

promoters and supports HBV replication in non-hepatic cells (9, 28). FoxA2 was shown

395

to inhibit HBV replication posttranscriptionally in hepatoma cells and transgenic mice (9,

396

10). In this study, we demonstrated that, another LETF, HNF6 could inhibit expression of

397

HBV secreted antigens and DNA replication. The overexpression of HNF6 reduced,

398

whereas the suppression of HNF6 expression increased the levels of HBV transcripts and

20

399

proteins (Fig. 1 and 3). Importantly, the HNF6-mediated HBV suppression was a

400

posttranscriptional event via accelerating the decay of pgRNA in the nucleus (Fig. 6 and

401

7). The role of HNF6 in HBV gene expression was also confirmed in vivo using a mouse

402

model, in which the overexpression of HNF6 lead to markedly reduction of HBV proteins

403

and DNA in the mice sera and liver (Fig. 9).

404 405

Regulation of HBsAg expression is crucial for the pathogenesis of HBV, as the envelope

406

proteins is essential for the viral particle formation, infectivity and immune response (2,

407

4). In the present study, an approximately 10-fold inhibition on HBsAg expression has

408

been observed upon HNF6 overexpression in mice (Fig. 9B). By performing reporter

409

assays, we demonstrated that the activity of SP2 promoter is inhibited by HNF6 (Fig. 4A).

410

Further analysis showed that the region from 3009-3019 of HBV genome is responsible

411

for the HNF6-mediated SP2 promoter inhibition through an indirect manner (Fig. 5).

412

However, HNF6 does not bind directly to the SP promoter region (nt 3009-3019) (Fig.

413

5D), and this suggest that HNF6 may function through some undefined factors that could

414

bind directly to the SP2 promoter. As no regulatory protein were previously reported to

415

bind with this region, we analyzed the transcription factors that can potentially bind to

416

this region by a bioinformatic method (MatInspector). Potential binding sites could be

417

predicted for four cellular factors: glial cells missing homolog 1, heat shock factor 1,

418

Kruppel-like factor (KLF15), and sterol regulatory element binding protein. Among these

419

factors, KLF15 has been shown to activate the SP2 promoter of HBV in a previous study,

21

420

but the precise binding site of KLF15 has not been defined (29). Therefore, it is

421

interesting to investigate whether KLF15 binds to this region and whether HNF6 can

422

suppress the KLF15-mediated SP2 activation in the future work. Moreover, the S mRNA

423

(2.1kb RNA) driven by SV40 or CMV promoter were also inhibited by HNF6

424

overexpression (Fig. 6), which suggests that both transcriptional and posttranscriptional

425

mechanisms may account for the profound suppression activity of HNF6 on HBsAg

426

expression synergistically.

427 428

By using the pSV40-HBV and the pCMV-HBV constructs, the HNF6-mediated HBV

429

pgRNA inhibition was shown to be independent of the nature of HBV promoters (Fig. 6).

430

Therefore, it is likely that HNF6 might influence the transcriptional elongation or the

431

stability of HBV RNAs. By using the tet-controlled system, we demonstrated that HNF6

432

could promote the degradation of HBV pgRNA, which is the template for viral reverse

433

transcription. Since HNF6 does not possess RNA binding or nuclease activity, it is

434

reasonable to hypothesize that a downstream effector accounts for the HNF6-mediated

435

HBV pgRNA decay. It has been shown that La protein and zinc finger antiviral protein

436

(ZAP) can regulate HBV RNA stability through directly binding to the their RNA target

437

via recognition sites (25, 30). La is capable of binding HBV RNAs and protecting them

438

from endonuclease cleavage. However, our results showed that the HNF6-mediated HBV

439

RNA decay is independent of La. ZAP binds ZAP responsive element in HBV RNA and

440

recruits exosome or other factors resulting in HBV RNA degradation. ZAP is not likely

22

441

the downstream effector of HNF6, for ZAP reduces HBV transcripts levels both in

442

hepatic and non-hepatic cell lines, which is different from the hepatocyte-specific

443

inhibition manner by HNF6 (30). Therefore, there might be other host factors, which

444

serve as the downstream effectors of HNF6, directly regulating the stability of HBV

445

RNAs. Actually, a previous report has showed that Myd88 promotes the decay of HBV

446

pgRNA in hepatocyte-specific manner through a cryptic effector other than the La antigen

447

(31). Although these data have demonstrated that HNF6 could accelerate the decay of

448

HBV pregenomic RNA, the possibility that HNF6 affects HBV RNA elongation cannot

449

be excluded. Therefore, more studies are required to explore this possibility.

450 451

As a transcriptional factor, HNF6 could directly modulate the promoter activity of its

452

target genes. HNF6 has also been shown to regulate some gene expression via interaction

453

with other cellular factors (32). The C-terminal CUT and Homeo domain of HNF6 is

454

responsible for the promoter binding and protein interacting activity, while the N terminal

455

part was shown to be involved in transcriptional activation of some promoters (27). Using

456

serial deletion mutants of HNF6, we identified that the N-terminal region is critical for

457

the inhibitory effects on HBV RNA. The N-terminal region alone was able to reduce the

458

level of HBV RNA (Fig. 8). Hence, the direct transcriptional regulatory activity of HNF6

459

was not required for the HNF6-mediated pgRNA inhibition, while the protein interaction

460

activity of the N-terminus might be required.

461

23

462

HNF6 regulates various important cellular functions, including liver and pancreas

463

development, differentiation, cell proliferation, and metabolisms (13). The expression of

464

HNF6 shows a female-predominant manner and controlled by GH, whose secretion is

465

pulsive in male mice, while constant in female mice (17). HNF6 was believed to be the

466

dominant effector for GH stimulated pathways (16, 33). Previous report showed that

467

female-specific expression of CYP2C12 attributes to the regulation of GH-HNF6

468

pathway (17). Interestingly, the gender disparity of HBV has also been well documented.

469

The male HBV carriers usually have higher viral load and higher possibility progressing

470

to HCC than those of the female carriers (34, 35). Hence, it is reasonable to speculate that

471

the GH/HNF6 axis might also partly account for the gender dimorphism of HBV.

472

However, more studies are warranted to demonstrate the role of HNF6 in the sex disparity

473

of HBV gene expression and replication.

474 475

Acknowledgments

476

This study was supported by the National Basic Research Program of China

477

(#2010CB911800) and the National Science Foundation of China （#31221061）. D.G. is

478

also supported by Hubei Province's Outstanding Medical Academic Leader Program. We

479

also thank Zhang Hui, Li Shun and Liu Ye for technical assistance, Ma Chunqiang, Su

480

Ceyang, Li Shun, Chen Shiyou and Chen Shuliang for meaningful discussion.

481 482

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Figure Legends

577

Fig. 1. HNF6 inhibits HBV gene expression and replication in hepatoma cells. HepG2

578

cells were transfected as indicated and harvested 48 hours after transfection (A, B, D, and

579

E) or 96 hours post transfection (C and F). (A)The expression of HNF6 was determined

580

by Western blot using an HA antibody. (B) Northern blot analysis of HBV transcripts.

581

The rRNA (18S and 28S) was presented as loading controls. (C) Southern blot analysis of

582

HBV DNA. RC, relaxed circular DNA; RI, replication intermediate DNA. The levels of

583

(D) HBsAg, (E) HBeAg and (F) HBV-DNA in the culture medium were measured by

584

ELISA and qPCR, respectively. The levels of HBsAg, HBeAg and extracellular

585

HBV-DNA, have been normalized against β-gal activities. Huh7 (G) and HEK 293T (H)

586

were transfected with the indicated amounts of plasmids. 48 hours post transfection, total

587

cellular RNA were extracted and analyzed by Northern blotting analysis. The data are

588

average of three independent experiments and statistically analyzed with a t test. * p