Indoleamine 2, 3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B

38 Pages·2010·2.92 MB·English

JVI Accepts, published online ahead of print on 17 November 2010
J. Virol. doi:10.1128/JVI.01998-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Indoleamine 2, 3-dioxygenase mediates the antiviral effect of gamma interferon against
hepatitis B virus in human hepatocyte-derived cells
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Richeng Mao1,2, Jiming Zhang2, Dong Jiang1§, Dawei Cai3, Jessica M. Levy3, Andrea Cuconati3, nlo
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Timothy M. Block1,3, Ju-Tao Guo1*, and Haitao Guo1* e
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1Institute for Biotechnology and Virology Research, Department of Microbiology and p:
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Immunology, Drexel University College of Medicine, 3805 Old Easton Rd, Doylestown, PA, i.
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18902. 2Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Wulumuqi
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Middle Rd, Shanghai, 200040 China. 3Institute for Hepatitis and Virus Research, Hepatitis B /

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Foundation, 3805 Old Easton Rd, Doylestown, PA, 18902. N
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Running head: IDO inhibits HBV 1
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*Corresponding authors, mailing address: g
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Haitao Guo, Ph.D. Institute for Biotechnology and Virology Research, Department of
Microbiology and Immunology, Drexel University College of Medicine, 3805 Old Easton Road
Doylestown, PA 18902. Phone: [phone]. Fax: [phone]. E-mail:
[email protected]
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Ju-Tao Guo, Drexel Institute for Biotechnology and Virology Research, Department of
Microbiology and Immunology, Drexel University College of Medicine, 3805 Old Easton Road
Doylestown, PA 18902. Phone: [phone]. Fax: [phone]. E-mail: Ju-
[email protected].
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§Current address: Peking University People’s Hospital, Hepatology Institute, 11 Xizhimen South o
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Street, Beijing 100044, China lo
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Interferon-alpha (IFN-αααα)))) is an approved medication for chronic hepatitis B.
Gamma Interferon (IFN-γγγγ) is a key mediator of host innate and adaptive antiviral
immunity against hepatitis B virus (HBV) infection in vivo. In an effort to elucidate the
antiviral mechanism of these cytokines, 37 IFN-stimulated genes (ISGs), which are highly
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inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon o
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overexpression in human hepatoma cells. One ISG candidate, indoleamine 2, 3-dioxygenase lo
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(IDO), an IFN-γγγγ-induced enzyme catalyzing tryptophan degradation, efficiently reduced e
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the level of intracellular HBV DNA, without altering the steady state level of viral RNA. o
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Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV
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replication, and tryptophan supplementation in culture completely restored HBV /
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replication in IDO-expressing cells, indicating that the antiviral effect elicited by IDO is s
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mediated by tryptophan deprivation. Interestingly, IDO mediated tryptophan deprivation o
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preferentially inhibited viral protein translation and genome replication, but did not o
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significantly alter global cellular protein synthesis. Finally, tryptophan supplementation o
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was able to completely restore HBV replication in IFN-γγγγ, but not IFN-αααα, treated cells,
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which strongly argues that IDO is the primary mediator of IFN-γ elicited antiviral response
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against HBV in human hepatocyte-derived cells. 2
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Hepatitis B virus (HBV) is a hepatotropic virus in the family of Hepadnaviridae (18, 56)
which has infected two billion people worldwide. Although most infected adults can resolve the
infection, approximately 5-10% adulthood infections and more than 90% of neonatal infections
become life-long persistent, which causes a significant public health burden currently affecting
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more than 350 million individuals worldwide (36, 41). Patients with chronic hepatitis B carry a
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high risk of cirrhosis and primary hepatocellular carcinoma (5, 36). n
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It is generally believed that the outcome of HBV infection and disease pathogenesis are d

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primarily determined by the host adaptive antiviral immune response (9). The resolution of HBV m

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infection is associated with a robust, polyclonal viral antigen specific cytotoxic T lymphocyte tt
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(CTL) response that kills and/or noncytopathically cures virally infected hepatocytes. The latter jv
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effect is primarily mediated by inflammatory cytokines, including gamma interferon (IFN-γ) and s
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tumor necrosis factor-alpha (TNF-α) (17-18, 20-21). Interestingly, although it has been shown rg
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that HBV can evade host innate immunity surveillance, as evidenced by its failure to induce the n

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expression of interferon-stimulated genes (ISGs) in the liver of chimpanzees during the early v
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phase of infection (66), the virus is sensitive to IFN-α/β induced antiviral response in transgenic b
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mice and in people (30, 49). For example, treatment of chronic hepatitis B with pegylated IFN-α 1
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for 48 weeks can achieve sustained virological response in 30-40 % of the treated patients (29). 0
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In efforts to elucidate the antiviral mechanism of IFNs, it has been demonstrated that y

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IFN-α and IFN-γ-induced antiviral responses either prevent the formation of pregenomic (pg) e
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RNA–containing capsids or promote the decay of replication competent nucleocapsids in murine
hepatocytes in culture and in transgenic mice in vivo (3, 67-68). Recently, type III IFN (IFN-λ)
has also been shown to inhibit HBV replication in cultured murine hepatocytes through a similar
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mechanism, but with a weaker antiviral activity in transgenic mice (49, 54). Ironically, IFN
treatment of human hepatoma-derived cells usually elicits no or a weaker antiviral response
against HBV. Moreover, it remains unclear whether IFNs inhibit HBV replication in human
hepatoma cells by targeting viral RNA transcription and/or disrupting viral DNA replication
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complexes (49, 53, 62).
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To further elucidate the mechanism of IFNs against HBV in human hepatocyte-derived lo
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cells, we first demonstrated that IFN-α and IFN-γ did not alter the steady state level of viral d

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RNA, but efficiently reduced the level of intracellular HBV DNA in HepG2-derived stable cell m

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lines supporting HBV replication. To determine IFN-induced cellular proteins that mediate the tt
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antiviral response of the cytokines, we systematically examined 37 ISGs that are inducible in jv
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hepatocytes by IFN-α and/or IFN-γ for their ability to inhibit HBV replication upon m
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overexpression in HepG2 cells. We identified two IFN-induced cellular enzymes, APOBEC3G rg
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and indoleamine 2, 3-dioxygenase (IDO), which significantly reduced viral DNA, but did not n

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alter the level of viral RNA. Further mechanistic analyses provided strong evidence suggesting v
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that the IDO elicited antiviral response against HBV depends on induction of tryptophan b
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deprivation, which preferentially inhibits viral protein translation, but does not significantly alter 1
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global cellular protein synthesis in HepG2 cells. Intriguingly, we were able to show that 0
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tryptophan supplementation completely restored HBV replication in cells treated with IFN-γ, but b
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not IFN-α. Our findings thus favor the hypothesis that IDO is the primary mediator of the IFN-γ u
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elicited antiviral response against HBV in human hepatocyte-derived cells. These results may
shed light on the mechanism by which CTL and IFN-γ-mediated immune response to
noncytolytically control HBV infection.
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MATERIALS AND METHODS
Cell cultures and drugs. HepG2 cells were obtained from ATCC and maintained in
DMEM/F12 medium (Mediatech) supplemented with 10% fetal bovine serum, 100 U/ml
penicillin and 100 µg/ml streptomycin. HepDE19 cells were maintained in the same way as
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HepG2, but with the addition of 1 µg/ml tetracycline and 400 µg/ml G418 (23). To initiate HBV
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replication in HepDE19 cells, tetracycline was withdrawn from the culture medium and the cells lo
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were cultured for the indicated time period. Recombinant human IFN-α2a and IFN-γ were d

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purchased from PBL Biomedical Laboratories. L-tryptophan (L-Trp) and 1-methyl-L-trp (1-MT) m

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were purchased from Sigma-Aldrich. tt
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Plasmid construction. The construction strategy of plasmids expressing individual i.
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human ISGs, including IDO (listed in table 1), was described in previous reports (33-34), except
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for inducible nitric oxide synthase (iNOS). The iNOS gene was PCR amplified from hep-iNOS /

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(15) (a gift of Dr. David Geller, University of Pittsburgh) and cloned into vector pcDNA5 N
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(Invitrogen) restricted with AflII and NotI. All the ISGs contain a FLAG tag sequence fused to e
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the N-terminus and their expression is under the control of a cytomegalovirus (CMV) immediate e
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early (IE) promoter. The enzymatically inactive mutant of IDO, IDOH346A (37), was constructed 7
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by introducing a single amino acid mutation into the IDO expression plasmid with the 1
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QuikChange® II Site-Directed Mutagenesis Kit (Stratagene). To construct an HBV core promoter by

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(Cp) reporter plasmid, a fragment covering the HBV enhancer II (EnII) and Cp region (nt 1400- e
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1902, genotype ayw, Genbank accession number V01460) was PCR amplified from plasmid
pHBV1.3 (25) and inserted into the SacI and HindIII restriction sites in pGL3-Basic vector
(Promega). The generated plasmid was designated EnII/Cp-Luc, in which the expression of
firefly luciferase reporter gene is governed by the HBV core promoter. The primer sequences
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used in this study are available upon request. The sequences of all the DNA clones were
confirmed by sequencing.
Cell transfection and cytotoxicity assay. Cells were seeded in a collagen coated 6-well-
plate at a density of 1.2 x 106 cells per well in antibiotics-free complete DMEM/F12 medium.
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After 6 hours, each well was transfected with a total of 4 µg plasmid with Lipofectamine 2000
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(Invitrogen) by following the manufacturer’s directions. Transfected cells were harvested at the n
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indicated time points. Cell viability was determined with the CellTiter 96® AQ Non- d
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Radioactive Cell Proliferation Assay (Promega), following the manufacturer’s instructions. r
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Reporter assay. HepG2 and HepDE19 cells were transfected using Lipofectamine 2000 tt
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(Invitrogen). For each transfection, 1 × 104 cells were cotransfected with 200 ng of plasmid /jv
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EnII/Cp-Luc and 4 ng of plasmid pRL-CMV (Promega). Sixteen hours after transfection, cells s
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were left untreated or treated with 1,000 IU/ml IFN-α2a or 100 ng/ml IFN-γ for 4 days, with r
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treatment repeated every other day. Cells were then harvested and lysed. Luciferase activities in on

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cell lysates were assayed using a dual luciferase assay system (Promega) and measured by o
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TopCount NXT.
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Viral nucleic acid analysis. Intracellular HBV core DNA and total cellular RNA were 1
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extracted as described previously (22-23). For Core DNA analysis, one third of the DNA sample 2
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from each plate was resolved by electrophoresis into a 1.5% agarose gel and blotted onto
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Hybond-XL membrane (GE Healthcare). For RNA analysis, ten microgram of total cellular RNA g
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was resolved in 1.5% agarose gel containing 2.2 M formaldehyde and transferred onto Hybond- t
XL membrane in 20× SSC buffer. Membranes were probed with either an α-32P-UTP (800
Ci/mmol, Perkin Elmer) labeled minus or plus strand specific full-length HBV riboprobe and
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exposed to a phosphorimager screen. Hybridization signals were quantified with QuantityOne
software (Bio-Rad).
RT-PCR analysis of ISG expression. HepG2 cells were left untransfected or transfected
with pHBV1.3. Sixteen hours later, cells were then left untreated or treated with 1,000 IU/ml of
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IFN-α or 100 ng/ml IFN-γ for 48 hours and harvested. Total cellular RNA was extracted with o
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TRIzol reagent by following the manufacturer’s directions (Invitrogen). The first strand cDNA lo
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was synthesized with oligo-(dT) primer and Superscript III DNA polymerase (Invitrogen). To
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quantify mRNAs for ISGs and β-actin, 0.5 µl of each cDNA reaction mixture was amplified for o
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25 cycles with respective forward and reverse primers (sequence available upon request). PCR t
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products were resolved by electrophoresis in a 1% agarose gel and stained with ethidium /jv
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bromide. s
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Protein analysis. For protein labeling, cells in one well of a 6-well-plate were rg
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cotransfected with 2 µg of plamids pHBV1.3 and 2 µg of control vector or plasmid expressing n

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IDO. Cells were washed twice with warm PBS and labeled at 37 oC for 1 hour at various time v
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points post-transfection with 35S-Methonine (Perkin Elmer) to a final concentration of 100 b
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µCi/ml. Cells were then lysed in 1 ml of lysis buffer containing 10 mM Tris-HCl, pH 8.0, 10 mM 1
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EDTA, 1% NP40, and 1 × protease inhibitor cocktail (Qiagen). Thirty microliters of the clarified 0
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cell lysate was directly mixed with 10 µl of 4 × Laemmli buffer and subjected to sodium dodecyl b
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sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a PVDF u
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membrane (Millipore). The remaining cell lysate was subjected to immunoprecipitation assay. t
Briefly, 500 µl of cell lysate was mixed with 20 µl of anti-FLAG® M2 affinity gel (Sigma) or
protein A/G plus beads (Santa Cruz) that were preabsorbed with antibodies against HBcAg
(Dako), respectively. The mixtures were incubated at R.T for 2 hours. Beads were washed four
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times with TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and boiled in 20 µl 1 ×
Laemmli buffer. The clarified samples were subjected to a 12% SDS-PAGE and transferred onto
a PVDF membrane (Millipore). The membranes were exposed to a phosphorimager screen and
radioactive signals were scanned and quantified with QuantityOne software (Bio-Rad).
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For Western blot analysis, cells in one well of a 6-well-plate were washed once with PBS
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buffer and lysed in 300 µl of 1× Laemmli buffer. Thirty microliters of the cell lysate was n
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resolved in a 10% SDS-PAGE and proteins were transferred onto an Immobolin™ PVDF-FL
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membrane (Millipore). The membranes were blocked with Western Breeze blocking buffer r
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(Invitrogen) and probed with antibodies against FLAG-tag (Sigma, F1804), IDO (Santa Cruz, h
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SC-25808), TDO (a gift of Dr. Christine Miller, John Hopkins University) (44), HBcAg (24), :/
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HBsAg (Fitzgerald, 20-HR20) and β-actin (Chemicon, MAB1501R). Bound antibodies were a
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revealed by IRDye secondary antibodies and visualized using the Li-COR Odyssey system. .o
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RESULTS
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IFN-αααα and IFN-γγγγ inhibit HBV replication in human hepatocyte derived cells. There b
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are several reports demonstrating that both IFN-α and IFN-γ are able to inhibit HBV replication 1
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in human hepatoma derived cell lines, such as HepG2 and Huh7. However, it remains 0
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controversial whether the cytokine-induced antiviral response primarily targets viral RNA or b
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DNA biosynthesis or degradation (28, 53). We reasoned that some of the discrepancy in the u
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previous reports might be related to the different cell culture systems employed in their studies. t
Therefore, we set out to compare, side by side, the effects of IFN-α and IFN-γ on the steady-state
levels of intracellular HBV RNA and DNA in HBV-replicon plasmids transiently transfected
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HepG2 cells and a HepG2-derived HBV replicating stable cell line, specifically HepDE19 cells
(23).
Interestingly, as shown in Fig. 1, while both IFN-α and IFN-γ efficiently reduced HBV
pgRNA and surface RNAs in HepG2 cells transiently transfected with plasmids transcribing the
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viral pgRNA under the direction of either the HBV authentic core promoter (pHBV1.3) or a w
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CMV-IE promoter (pCMVHBV) (Fig. 1A and B, upper panels), neither IFN-α nor IFN-γ a
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affected the levels of HBV RNAs in HepDE19 cells (Fig. 1C, upper panel). However, the d

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cytokines efficiently reduced HBV DNA in both transiently and stably transfected HepG2 cells m

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in a dose-dependent manner (Fig. 1A, B and C, lower panels). Such observation is consistent t
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with previous reports showing IFN reduced HBV promoter activity in the reporter gene jv
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transfection assay (62), but not in a stable cell line harboring the integrated tandem HBV dimer, m
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in which the viral RNA expression is under the control of authentic viral promoters (28). These rg
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results thus imply that IFN-induced antiviral response selectively inhibits the transcription of n

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HBV RNA from input plasmids in transiently transfected cells, but not from the integrated HBV v
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transgene in the stable cell line. To further confirm this notion and rule out the possibility that the b
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observed difference on HBV RNA level is due to different cell line (HepG2 versus HepDE19), 1
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we performed reporter assays by co-transfection of plasmids encoding HBV core promoter- 0
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directed firefly luciferase (EnII/Cp-Luc) and CMV-IE promoter-directed Renilla luciferase (pRL- b
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CMV) into either HepG2 cells or HepDE19 cells and treated with IFN-α and IFN-γ, respectively. u
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As shown in Fig. 1D, the results indicate that the IFNs significantly inhibited HBV core and
CMV IE-promoter driven transcription in both HepG2 and HepDE19 cells at a similar extent,
suggesting that the effect of IFNs on transcription is a specific phenomenon for transfected
plasmid-based transcription template.
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