STAT1-Independent Cell Type-Specific Regulation of Antiviral APOBEC3G by IFN- α

This information is current as Phuong Thi Nguyen Sarkis, Songcheng Ying, Rongzhen Xu of October 2, 2021. and Xiao-Fang Yu J Immunol 2006; 177:4530-4540; ; doi: 10.4049/jimmunol.177.7.4530 http://www.jimmunol.org/content/177/7/4530 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

STAT1-Independent Cell Type-Specific Regulation of Antiviral APOBEC3G by IFN-␣1

Phuong Thi Nguyen Sarkis,* Songcheng Ying,† Rongzhen Xu,† and Xiao-Fang Yu2*†

APOBEC3G (A3G) has broad antiviral activity against and hepatitis B . However, the role of IFNs in regulating A3G during innate immunity has not been established. In this study, we show that the A3G is uniquely regulated by IFNs in a cell type-dependent manner. A3G was up-regulated by IFN-␣ in liver cells and , but not in T lymphoid cells or epithelial 293T cells. In contrast, other IFN-␣-stimulated such as dsRNA-activated kinase were induced in all these cells, suggesting additional cellular factors may regulate IFN-␣-induced A3G expression. Consistent with this idea, IFN-␣-medi- ated induction of A3G, but not other IFN-␣-stimulated genes, was potently inhibited by the drug Rottlerin, through a mechanism independent of STAT1 activation. The canonical IFN-␣-mediated pathway of gene transcription requires both STAT1 and STAT2. Surprisingly, induction of A3G was STAT1 independent, but STAT2 dependent in liver cells. However, STAT1 signaling was functional and required for IFN-␥ induction of A3G in these cells. Our results indicate that A3G may participate in antiviral Downloaded from cellular defenses through a novel IFN-mediated signaling pathway. The Journal of Immunology, 2006, 177: 4530–4540.

he APOBEC3G (A3G) is a member of the APOBEC fam- whether A3G participates in IFN-mediated host defenses is un- ily of with homologous do- clear. One previous study found that A3G was induced by PMA, mains (1). Although its cellular target(s) is not known, but not induced by IFNs in an immortalized CD4ϩ line H9

T http://www.jimmunol.org/ A3G is a potent antiviral protein that can suppress HIV type 1 (23). However, recent studies reported that A3G could be induced (HIV-1),3 hepatitis B virus (HBV), and endogenous retro-elements by IFNs in liver cells (24, 25) and macrophages (26). The effects (2–8). The antiviral mechanism of A3G against HIV-1 has been of IFNs on A3G transcription in primary CD4ϩ T cells have not well studied. In the absence of the viral Vif protein, the A3G cy- been reported. In the present study, we surveyed the ability of IFNs tidine deaminase converts cytidines to uridines in single-stranded to up-regulate A3G in various cells, including various primary viral cDNA during reverse transcription, resulting in lethal hyper- cells (hepatocytes, macrophages, and CD4ϩ T cells) and cell lines mutation of the virus genome (9–13). The HIV-1 Vif protein, how- (liver carcinoma cells, H9 cells, and 293T cells). All of these cells, ever, can degrade A3G through a proteasome-dependent pathway except T cells, up-regulated A3G mRNA in response to IFN-␣. (14–20) involving the Cullin5-containing E3 (14). ␥

Only liver cells consistently responded to IFN- . by guest on October 2, 2021 By targeting A3G for degradation in the virus-producing cell, Vif IFNs are capable of inducing hundreds of genes, some of which is able to prevent A3G molecules from incorporating into virions, may have the potential to affect the viral life cycle. Therefore, where they would otherwise be carried into the newly infected cell understanding how A3G might be uniquely regulated would help to inhibit productive infection. Although HBV is a DNA virus, it to distinguish its role in antiviral defense from that of other IFN- replicates through reverse transcription and can likewise be tar- stimulated genes (ISG). IFN-␣ and IFN-␥ signal through separate geted by A3G (21, 22). type I and type II IFN receptors, respectively, resulting in phos- Although many studies have focused on the antiviral effects and posttranslational regulation of A3G, little is known about how it is phorylation of the associated Jak. (For reviews of Jak/STAT sig- transcriptionally regulated. It is not known whether A3G may be naling pathways, see references 27–32). The result is a cascade of induced in vivo in response to inflammation or cytokines, and signaling events through the STAT family of proteins to induce transcriptional activation of ISG. The classical models assert that IFN-␣ induces transcription through a STAT1:STAT2 heterodimer *Department of Molecular Microbiology and Immunology, Johns Hopkins in complex with the IFN regulatory factor (IRF)-9 , which Bloomberg School of Public Health, Baltimore, MD 21205; and †Zhejiang University, Zhejiang, China binds to an IFN-stimulated response element (ISRE) in the pro- ␣ ␥ Received for publication February 24, 2006. Accepted for publication July 14, 2006. moter of IFN- -responsive genes. The IFN- pathway uses the STAT1 homodimer without a cofactor, which binds to the ␥-acti- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance vated sequence in gene promoters. Many ISG are inducible by with 18 U.S.C. Section 1734 solely to indicate this fact. either IFN-␣ or IFN-␥ exclusively, but some genes (e.g., low mo- 1 This work was supported by a grant from the National Institutes of Health lecular mass polypeptide (LMP)2) are inducible by both cytokines (AI062644), a grant from the Johns Hopkins Center for AIDS Research, and funding ␣ from the National Science Foundation of China (NSFC-30425012) and Cheung Kong (29, 33). In our study, we showed that IFN- uses a novel STAT1- Scholars Program Foundation of the Chinese Ministry of Education (to X.-F.Y.). independent pathway to up-regulate A3G, along with certain other 2 Address correspondence and reprint requests to Dr. Xiao-Fang Yu, Department of ISG, in liver cells. In addition, transcriptional activation of A3G Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Pub- was uniquely sensitive to the drug Rottlerin in liver cells through lic Health, 615 North Wolfe Street, Room E5148, Baltimore, MD 21205. E-mail address: [email protected] a mechanism independent of STAT1 activation. Although STAT1- ␥ 3 Abbreviations used in this paper: HIV-1, HIV type-1; HBV, hepatitis B virus; IRF, independent pathways of IFN- transcriptional activation are IFN regulatory factor; ISG, IFN-stimulated gene; ISGF3, ISG factor 3; ISRE, IFN- known, to our knowledge this will be the first report of a STAT1- stimulated response element; LMP, low molecular mass polypeptide; PKC, protein ␣ kinase C; PKR, dsRNA-activated protein kinase; qRT-PCR, quantitative real-time independent pathway of IFN- -mediated transcriptional activation RT-PCR; siRNA, small interfering RNA. of antiviral genes. Therefore, our results demonstrate that A3G

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 4531 may exert its antiviral activities through a novel IFN-mediated Immunoblot assays signaling pathway. In preparation for immunoblot assays, cells were lysed in PhosphoSafe extraction buffer (Novagen), diluted in 1ϫ loading buffer (0.08 M Tris (pH Materials and Methods 6.8), with 2.0% SDS, 10% glycerol, 0.1 M DTT, and 0.2% bromphenol Cells blue), and boiled for 5 min. Equal amounts of cell lysates were separated on SDS-PAGE gels and transferred onto nitrocellulose membranes using a Freshly isolated primary hepatocytes from anonymous donors were ob- semidry apparatus (Bio-Rad). Membranes were probed with various pri- tained from BD Biosciences. Hepatocytes were received within 24–48 h of mary Abs against the proteins of interest; secondary Abs were alkaline isolation as adherent cultures in 6-well plates in Hepatostim medium (BD phosphatase-conjugated anti-rabbit IgG and anti-human IgG (Jackson Im- Biosciences) and used immediately for induction studies. Hep3B, HepG2 munoResearch Laboratories). Staining was conducted with 5-bromo-4- (American Type Culture Collection), and Huh7 (a gift from C. Rice, The chloro-3-indolyl phosphate and NBT solutions prepared from chemicals Rockefellar University, New York, NY) are hepatocellular carcinoma cell obtained from Sigma-Aldrich. lines. The 293T cells were obtained from American Type Culture Collec- tion. The U3A are STAT1-negative and U6A are STAT2-negative cells RNA interference derived from the parental 2fTGH cells (a gift from G. Stark, Cleveland RNA interference against STAT1, STAT2, IRF-9, STAT3, and PKC-␦ was Clinic Foundation, Cleveland, OH). All cell lines were maintained in conducted using SMARTpool siRNAs, which are predesigned pools of DMEM (Invitrogen Life Technologies) supplemented with 10% FBS. Pri- ϩ four duplexed siRNAs targeting a single gene (Dharmacon). Cells were mary CD4 T cells were purified from freshly isolated PBMC by incuba- transfected with siRNA pools at a total final concentration of 100 nM using tion with CD4-conjugated magnetic microbeads (Miltenyi Biotec), accord- Lipofectamine 2000 (Invitrogen Life Technologies). siCONTROL nontar- ing to the manufacturer’s instructions, and cultured in RPMI 1640 with geting siRNA 2 (Dharmacon) was used as a control. Ninety-six hours after 10% FBS. To obtain macrophages, freshly isolated PBMC were plated in transfection, IFNs were added to cells for a period of 8 h before processing 7 6-well plates overnight at 2 ϫ 10 cells/ml in RPMI 1640 with 10% FBS, for mRNA detection by qRT-PCR. after which nonadherent cells were removed and the medium was replaced Downloaded from every 2 days. Macrophages, differentiated via adherence to the plastic, Results were used on day 12 after isolation for induction studies. Induction of A3G by IFN is donor and cell-type dependent Cytokines, Abs, and inhibitors Recent studies reported that A3G is inducible by IFN-␣ in liver All cytokines and inhibitors were obtained from EMD Biosciences. IFN-␣ cells (24, 25) and macrophages (26), but not in immortalized ϩ and IFN-␥ were dissolved in PBS with 0.5% BSA (control medium) and CD4 T cells (23). The effects of IFNs on A3G transcription in stored in single-use aliquots at Ϫ70°C. Unless otherwise stated, IFN-␣ was primary CD4ϩ T cells and other cell types such as epithelial cells http://www.jimmunol.org/ ␥ used at 1000 IU/ml and IFN- at 10 IU/ml. In IFN induction experiments, have not been reported. In the present study, we tested the ability cells are treated with equal volumes of either IFN or control medium. The Ϫ of IFNs to up-regulate A3G in various primary cells (hepatocytes, following inhibitors were dissolved in DMSO, stored in aliquots at 20°C, ϩ and used at indicated final concentrations: Jak inhibitor I (5 ␮M), Go6983 macrophages, and CD4 T cells) and cell lines (liver carcinoma (1 ␮M), GF109203X (5 ␮M), Rottlerin (6 ␮M), and actinomycin D (5 cells, 293T cells, and H9 cells). Using a qRT-PCR method, we ␮g/ml). In experiments using these inhibitors, equivalent volumes of in- observed that A3G was up-regulated by both IFN-␣ and IFN-␥ in hibitor or DMSO control were used. Final concentrations of DMSO were primary hepatocytes from one donor (85) through a time course of Ͻ0.5%. Anti-A3G, from W. Greene (University of California, San Fran- cisco, CA) was obtained through the National Institutes of Health AIDS up to 24 h (Fig. 1A) and followed a similar kinetic to that of PKR Research and Reference Reagent Program, Division of AIDS, National (Fig. 1B), a known IFN-␣-inducible gene. IFN-␥ also induced by guest on October 2, 2021 Institute of Allergy and Infectious Diseases, (16). All other Abs were com- A3G (Fig. 1A) in the hepatocytes, but to a lesser extent. IFN- 701 mercially available: anti-STAT1, anti-STAT3, and anti-pSTAT1 Tyr induced expression of A3G protein in primary hepatocytes was (Santa Cruz Biotechnology); anti-STAT2, anti-pSTAT3 Tyr705, and anti- pSTAT1 Ser727 (BioSource International); anti-protein kinase C (PKC)-␦ also demonstrated by immunoblotting (Fig. 1C). This result is con- (Cell Signaling Technology); anti-IRF-9 (BD Biosciences); and anti- sistent with the previous studies that reported on IFN-␣ induction ribosome p19 (ImmunoVision). of A3G in primary hepatocytes (24). We asked whether there are differences in A3G expression among multiple individuals, and Quantitative real-time RT-PCR (qRT-PCR) more importantly, whether there are quantitative differences in the qRT-PCR was performed according to standard protocols (34). Briefly, levels of A3G after IFN-␣ treatment. Therefore, we compared the total RNA from cells was isolated using the RNeasy Mini Kit (Qiagen), A3G expression levels in control primary hepatocytes and cells according to the manufacturer’s instructions, including an on-column ␣ DNase digestion step using the RNase-free DNase Set (Qiagen). One-fifth treated with IFN- from four donors (78, 79, 80, and 85). The of the RNA was reverse transcribed using random primers and the Multi- comparative ⌬⌬CT method was used to calculate relative expres- scribe from the High Capacity cDNA Archive Kit sion using ␤-actin as a normalizing gene, and the lowest A3G- (Applied Biosystems). The cDNA was amplified using TaqMan Universal expressing cell sample in the group (control-treated hepatocytes PCR master mix (Applied Biosystems) and an ABI Prism 7000 sequence from donor 78) was set as the calibrator with an arbitrary unit of detection system (Applied Biosystems). The primers/probe sets were pre- designed TaqMan gene expression assays specific for A3G, dsRNA-acti- 1. A3G expression in other cells was expressed as relative fold vated protein kinase (PKR), IRF-1, ISG15, MX1, and LMP2 (assay iden- units above the calibrator. Some variation in A3G expression can tification numbers: Hs00222415_m1, Hs00169345_m1, Hs00233698_m1, be noted between the untreated cells, but a more marked variation Hs00192713_m1, Hs00182073_m1, and Hs00160610_m1, respectively). can be observed after IFN treatment (Fig. 1D). The range of A3G Amplification of target genes was normalized using amplification levels of ␤-actin as an endogenous control (human ACTB (␤-actin) endogenous mRNA expressed was between 9- and 38-fold above the calibrator control FAM/MGB probe; Applied Biosystems). The efficiency of the PCR after IFN-␣ treatment. In contrast, the range of PKR mRNA ex- was tested by amplification of the target from serially diluted cDNA gen- pressed was between 6- and 13-fold over the calibrator after IFN-␣ erated from reverse transcription of a stock set of human RNA. Data anal- treatment (Fig. 1E). Ϫ⌬⌬CT ysis and calculations were performed using the 2 comparative Although IFN-␣ could induce A3G mRNA (Fig. 2A) and A3G method, as previously described (34). Gene expression is expressed as fold protein (Fig. 2C) as well as PKR (Fig. 2B) in primary macrophages inductions of a gene measured in IFN-treated samples relative to samples ϩ treated with control medium (PBS 0.5% BSA). A Ͼ2-fold induction is from donor 1, A3G could not be induced in primary CD4 T cells considered positive. In experiments in which inhibitors were used, relative (Fig. 2D) from the same donor, while PKR (Fig. 2E) was posi- gene expression in IFN-treated samples was measured against control- tively induced. We consistently observed that A3G was not up- treated samples that were pretreated with the same inhibitor or DMSO ϩ control. In experiments with small interfering RNA (siRNA)-transfected regulated by IFN in primary CD4 T cells or whole PMBC from cells, relative gene expression in IFN-treated samples was measured several other donors (data not shown). IFN-␥ did not significantly against control-treated samples that were transfected with the same siRNA. induce A3G in macrophages from this particular donor (Fig. 2A). 4532 STAT1-INDEPENDENT REGULATION OF A3G BY IFN-␣ Downloaded from http://www.jimmunol.org/

FIGURE 1. Induction of A3G by IFN is donor dependent. A and B, Primary hepatocytes from donor 85 were treated with IFN-␣, IFN-␥, or control medium for 0, 8, 16, or 24 h, and then cells were collected to measure fold inductions of A3G or PKR by qRT-PCR. C, Primary hepatocytes from donor 85 were treated with IFN-␣, IFN-␥, or control medium for 24 h. A3G protein or ribosome p19 (loading control) was detected by immunoblotting of the cell lysates. D and E, Primary hepatocytes from four donors (78, 79, 80, and 85) were treated with control medium or IFN-␣ for 16 h. A3G mRNA and PKR mRNA were measured by qRT-PCR using the comparative ⌬⌬CT method and ␤-actin as a normalizing gene. Relative A3G or PKR expression in control- or IFN-treated cells was calculated by setting the A3G or PKR level in control cells from donor 78 (chosen as the calibrator because it expresses

lowest amount of A3G and PKR) as 1 and mRNA expression in other cells as relative fold units above the calibrator. by guest on October 2, 2021

However, it has been reported that A3G could be induced by for 1 h before addition of IFN-␣ inhibited IFN-mediated induction IFN-␥ in macrophages (26), and we also observed A3G induction of A3G (Fig. 3A). However, induction of other IFN-␣-responsive by IFN-␥ in one of the several donors that we tested genes such as PKR (Fig. 3B) and ISG15 (Fig. 3C) was not affected (data not shown). As previously reported (23, 24), we confirmed by Rottlerin. General PKC inhibitors (Go6983 and GF109203X) that IFN-␣ could not induce A3G in the H9 T cell line (Fig. 2F), did not inhibit A3G induction by IFN-␣ in Hep3B cells (Fig. 3A), but that it could in liver cell lines Hep3B, HepG2, and Huh7 (Fig. suggesting that none of the PKC isoforms participates in IFN-␣ 2G). Although IFN-␣ was generally more effective for inducing signaling in liver cells. To investigate whether PKC-␦ was actually A3G in primary hepatocytes (Fig. 1, A and D), IFN-␥ was more involved in the specific transcriptional activation of A3G, we effective for inducing A3G in two of the liver cell lines (Hep3B transfected Hep3B cells with PKC-␦ gene-specific siRNA (Fig. and HepG2) (Fig. 2H). As positive controls for IFN stimulation, 3D). Despite the fact that PKC-␦ was dramatically reduced in these we detected other known ISG such as PKR, which was mainly cells, induction of neither A3G, PKR, nor ISG15 genes by IFN-␣ induced by IFN-␣, and IRF-1, which was primarily induced by was affected, when compared with that in cells treated with control IFN-␥ (Fig. 2, G and H). siRNA (Fig. 3E). STAT1 was constitutively phosphorylated at Taken together, these results indicate that A3G regulation by Ser727 in Hep3B cells, but this phosphorylation was enhanced by IFNs is cell-type and donor dependent. The observed individual IFN-␣ treatment (Fig. 3F). Rottlerin also did not reduce the en- variation in IFN responsiveness in primary hepatocytes may ac- hanced phosphorylation of STAT1 at Ser727 by IFN-␣ (Fig. 3F). count for differences in viral pathogenesis and clinical outcomes in Therefore, the specific inhibition of IFN-␣-induced A3G mRNA HBV infection. expression by Rottlerin is apparently independent of PKC-␦ and STAT1 activation. A3G is uniquely regulated by a Rottlerin-sensitive target in To examine whether Rottlerin might affect the stability of A3G Hep3B liver cells mRNA after IFN-␣ induction, we treated Hep3B cells with IFN-␣ PMA induces A3G transcription in T cells through PKC-␣␤ acti- or control medium for 8 h, and then added Rottlerin or DMSO to vation (23). The PKC pathway also has been linked to the IFN the cultures with actinomycin D to stop transcription (Fig. 3G). pathway of transcription. The PKC-␦ isoform, in particular, was A3G mRNA levels were then measured in the IFN-␣-treated sam- reported to be the kinase responsible for phosphorylating STAT1 ples relative to the control samples at times up to 12 h after addi- at Ser727, a modification reportedly required for full transcriptional tion of Rottlerin or DMSO. The level of A3G mRNA was ex- activation of ISG (35). We observed that preincubation of Hep3B pressed as a relative percentage of the amount obtained after the cells with Rottlerin, reported to be a specific inhibitor of PKC-␦, 8-h incubation with IFN-␣ (time 0 in Fig. 3G), which was set at The Journal of Immunology 4533 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Induction of A3G by IFN is cell-type dependent. A and B, Primary macrophages from donor 1 were treated with IFN-␣, IFN-␥, or control medium for 0, 2, 4, 8, and 16 h, and then cells were collected to measure fold inductions of A3G or PKR by qRT-PCR. C, Primary macrophages from donor 1 were treated with IFN-␣, IFN-␥, or control medium for 24 h. A3G protein or ribosome p19 (loading control) was detected by immunoblotting of the cell lysates. D and E, Primary CD4ϩ T cells from donor 1 were treated with IFN-␣, IFN-␥, or control medium for 0, 2, 4, 8, and 16 h, and then cells were collected to measure fold inductions of A3G or PKR by qRT-PCR. F, H9 cells were treated with IFN-␣ or control medium for 8 h, and then cells were collected to measure fold inductions of A3G or PKR for qRT-PCR. Error bars, Fold inductions from triplicate cell samples. G and H, Liver cell lines HepG2, Hep3B, or Huh7 were treated with IFN-␣, IFN-␥, or control medium for 8 h, and then cells were collected to measure fold inductions of A3G, PKR, or .Off-scale; value ϭ 76 ,ء .IRF-1. Error bars, Fold inductions from triplicate cell samples

100%. A3G mRNA stability was not different between cells STAT1 knockdown did not block the IFN-␣-mediated induction of treated with Rottlerin or DMSO. Therefore, it is likely that Rot- A3G (Fig. 4B) or of certain other ISG such as PKR (Fig. 4C), tlerin interfered with IFN-␣-induced A3G (Fig. 3A) at the tran- ISG15 (Fig. 4D), or MX1 (Fig. 4E). However, induction of LMP2 scriptional level. by IFN-␣ was significantly inhibited by the STAT1 knockdown (Fig. 4F), suggesting that STAT1 activation by IFN-␣ was re- ␣ IFN- induction of A3G is independent of STAT1, but dependent quired for some genes in these liver cells. Induction of A3G, PKR, on STAT2 in Hep3B liver cells ISG15, or Mx1 by IFN-␣ was blocked by STAT2 knockdown (Fig. Our data to date have suggested a unique pathway of IFN induc- 4, B–F), indicating that STAT2, but not STAT1, remains critical tion of A3G. Therefore, we examined whether the induction of for the IFN-␣ induction of these genes in liver cells. A3G by IFNs was indeed dependent on the classical Jak/STAT To study whether STAT1 was indeed activated in these cells, we pathways that mediate transcriptional regulation of other well- treated the Hep3B liver cells with a general inhibitor of Jak, Jak known antiviral genes (27–31). The importance of STAT1 and inhibitor I, for 1 h before induction with IFNs for8hinthecon- STAT2 is well established in the literature for the IFN-␣ pathway tinuous presence of the drug. Phosphorylation of STAT1 at Tyr701 of transcription. Therefore, we transfected Hep3B cells with a non- by Jak is required for dimerization and transcriptional activation. targeting control siRNA or siRNA specific for STAT1 or STAT2. STAT1 Tyr701 phosphorylation was induced by IFN-␣ and was After 4 days, we treated the cells with either control medium, blocked by Jak inhibitor I (Fig. 4G). Inhibition of the Jak potently IFN-␣, or IFN-␥ for 8 h. The cells were then collected to measure blocked the induction of both A3G (Fig. 4H) and PKR (Fig. 4I)by gene expression by qRT-PCR and protein expression by immuno- IFN-␣, indicating that A3G required Jak for transcriptional acti- blotting. Both STAT1 and STAT2 proteins were efficiently and vation. Our data suggest that despite a functional mechanism of specifically reduced in siRNA-treated cells (Fig. 4A). Surprisingly, Jak-dependent activation of the STAT1 pathway in Hep3B cells, 4534 STAT1-INDEPENDENT REGULATION OF A3G BY IFN-␣ Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. A3G is uniquely regulated by a Rottlerin-sensitive target in Hep3B liver cells. A–C, Hep3B cells were treated with DMSO, Go6983, GF109203X (GFX), Jak Inhibitor I (Jak Inh I), or Rottlerin (Rot) for 1 h before treatment with IFN-␣ or control medium for 8 h. Relative mRNA expression of A3G, PKR, and ISG15 was measured by qRT-PCR. Induction of genes by IFNs is expressed as a fold induction of IFN-treated cells relative to control-treated cells. D, Hep3B cells were transfected with control or PKC-␦ siRNA for 4 days, and then treated with IFN-␣ or control medium for 8 h. The cells were then harvested to measure PKC-␦ protein or ribosome p19 as a loading control. E, One-half of the cells treated in D were collected for measurement of A3G, PKR, and ISG15 mRNA induction by qRT-PCR. F, Hep3B cells were treated with DMSO, Jak Inhibitor I (Jak), or Rottlerin (Rot) for 1 h. The cells were then treated with IFN-␣ for 1 h before collection for immunoblot analysis. Equal amounts of cell lysates were run in separate SDS-PAGE gels and transferred to three separate nitrocellulose membranes. Phosphorylated STAT1 proteins were detected using Abs against phospho-STAT1 Ser727 and phospho-STAT1 Tyr701 proteins. Total STAT1 protein was detected in the third membrane as a total protein-loading control. G, Hep3B cells were treated with IFN-␣ for 8 h, and then Rottlerin or DMSO control was added to the cultures with actinomycin D (transcription inhibitor). A3G induction was measured in the IFN-␣-treated samples over the control samples at times 0, 4, 8, and 12 h postaddition of Rottlerin or DMSO. Relative mRNA stability of A3G was expressed as a relative percentage of the amount obtained after the 8-h incubation with IFN-␣ (time 0 in Fig. 2G), which was set at 100%.

STAT1 itself was not required for IFN-␣-induced A3G expression. for8hinthecontinuous presence of the drug. IFN-␥ induced This observation was not specific to Hep3B cells, as we have also STAT1 Tyr701 phosphorylation in these cells, which was observed STAT1-independent, IFN-␣-induced A3G expression in blocked by Jak inhibitor I (Fig. 5A). Inhibition of Jak potently another liver cell line, Huh7 (data not shown). blocked the induction of A3G (Fig. 4B) by IFN-␥ similar to the IFN-␥-induced IRF-1 (Fig. 4C). After reducing STAT1 or ␥ IFN- induction of A3G is dependent on STAT1 in Hep3B STAT2 by transfecting Hep3B cells with gene-specific siRNA liver cells (Fig. 4A), we tested whether A3G or other ISG could be in- We next investigated whether STAT1 is functional and required duced. As expected, knockdown of STAT1, but not STAT2 for the IFN-␥ pathway. Unlike the IFN-␣ pathway of transcription, protein effectively blocked the induction of A3G (Fig. 5D) and the IFN-␥ pathway uses a STAT1 homodimer that forms upon the control gene IRF-1 (Fig. 5E) by IFN-␥. Therefore, STAT1 activation. We treated Hep3B liver cells with a general inhibitor of is functional and required for the IFN-␥ pathway of gene tran- Jak, Jak inhibitor I, or DMSO for 1 h before induction with IFNs scription in these cells. The Journal of Immunology 4535

FIGURE 4. IFN-␣ induction of A3G is in- dependent of STAT1, but dependent on STAT2 in liver cells. A, Hep3B cells were transfected with control, STAT1, or STAT2 siRNA. After 96 h, cells transfected with each type of siRNA were treated with IFN-␣, IFN-␥, or control medium for 8 h. Protein ex- pression of STAT1, STAT2, and ribosome p19 control was detected by immunoblotting of proteins from the same SDS-PAGE gel that were transferred to two membranes as identi- cal mirror images. B–F, Hep3B cells treated as in A were processed for qRT-PCR assay to measure induction of A3G, PKR, ISG15, MX1, and LMP2. Error bars, SDs of fold in- ductions from triplicate cell samples. G, Downloaded from Hep3B cells were treated with Jak inhibitor I (Jak) or DMSO for 1 h, and then treated with IFN-␣ or control medium for 1 h. Equal amounts of cell lysates were run on separate SDS-PAGE gels to detect pSTAT1 Tyr701 or total STAT1 proteins. H and I, Hep3B cells http://www.jimmunol.org/ were treated with Jak inhibitor I (Jak Inh I) or DMSO for 1 h, and then IFN-␣ or control me- dium was added to the culture for 8 h. Fold induction of A3G or PKR by IFN-␣ over con- trol cells was measured by qRT-PCR. Error bars, SDs of fold inductions from triplicate cell samples. by guest on October 2, 2021

IRF-9 is required for IFN-␣ induction of A3G and other ISG in STAT1 siRNA did not further decrease the up-regulation (Fig. 7, Hep3B cells D–H), further supporting that STAT1 is not necessary for the IFN It has been shown previously that STAT2 and IRF-9 can weakly induction of ISG in this cell line. bind DNA and mediate transcription in the absence of STAT1 (36, 37). Therefore, we tested the requirement for IRF-9 in the IFN-␣ STAT1 is required for both the IFN-␣ and IFN-␥ induction of induction of A3G and other ISG in Hep3B cells. We transfected ISG in 293T cells Hep3B cells with control, IRF-9, IRF-9 plus STAT1, or IRF-9 plus STAT2 siRNA for 4 days, and then treated the cells with control To determine whether our observation of the STAT1-independent medium or IFN-␣ for 8 h. In the absence of cytokine stimulation, IFN-␣-induced gene expression was unique to liver cells, we tested IRF-9 protein was not detected by immunoblotting, but highly in- whether STAT1 was required in 293T cells for the IFN-␣ pathway duced by IFN-␣ (Fig. 6A). In cells treated with IRF-9 siRNA, of transcription. We transfected 293T cells with control, STAT1, induction of IRF-9 protein by IFN-␣ was reduced (Fig. 6A). In or STAT2 siRNA for 4 days, and then treated the cells with either separate Western blot gels, STAT1 and STAT2 proteins were also IFN-␣ or IFN-␥ for 8 h. Both STAT1 and STAT2 proteins were confirmed to be reduced by the corresponding siRNA (Fig. 6, B efficiently and specifically reduced (Fig. 7A). Although A3G was and C). By qRT-PCR, we observed that cells treated with IRF-9 not inducible in this cell line (Fig. 7B), we observed that IFN-␣ siRNA reduced induction of A3G mRNA by IFN-␣ (Fig. 6D). This induction of other ISG such as ISG15 (Fig. 7C), MX1 (Fig. 7D), reduction of A3G transcription was similar in IRF-9 plus STAT1 and LMP2 (Fig. 7E) was inhibited by both STAT1 and STAT2 siRNA- or IRF-9 plus STAT2 siRNA-treated cells (Fig. 6D). siRNA. As observed in Hep3B cells (Fig. 5E), IFN-␥ induction of IRF-9 siRNA also reduced IFN-␣ induction of other ISG, such as IRF-1 (Fig. 7F) as well as LMP2 (Fig. 7G) was inhibited specif- PKR (Fig. 6E), LMP2 (Fig. 6F), ISG15 (Fig. 6G), and MX1 (Fig. ically by STAT1 siRNA and not STAT2 siRNA in 293T cells. 6H). IRF-9 siRNA did not completely inhibit the up-regulation of Therefore, STAT1 was required for both the IFN-␣ and IFN-␥ any ISG tested in this study, presumably because IRF-9 was min- induction of ISG in 293T cells, consistent with the classical model imally induced with IFN-␣. Combination treatment with IRF-9 and of IFN-signaling complexes. 4536 STAT1-INDEPENDENT REGULATION OF A3G BY IFN-␣

that H9 cells are relatively resistant to certain IFN-induced re- sponses (38–41), we observed normal IFN-␣-induced PKR ex- pression in H9 cells as well as primary CD4ϩ T lymphocytes (Fig. 2, E and F). This observation suggests at least some IFN-␣-medi- ated signaling pathways are functional in these T cells. It remains to be discovered why A3G could not be induced in T cells, al- though it is possible that a factor required for specific induction of A3G is absent in T cells. Liver cells and macrophages are primary targets of and HBV, respectively. It is therefore plau- sible that up-regulation of A3G by IFNs could contribute to innate in vivo antiviral defenses against HBV in liver cells and lentivi- ruses in macrophages. Although two recent studies had also reported that A3G could be induced by IFN-␣ in primary hepatocytes (24, 25), our study offered a quantitative comparative analysis of A3G induction by both IFN-␣ and IFN-␥ in multiple primary hepatocytes. We found that there was a wide variation in the level of A3G expressed after IFN-␣ treatment of hepatocytes from four individual donors (Fig.

1D). It is known that the abilities of spontaneous clearance of viral Downloaded from infection after HBV exposure differ significantly among various subjects (42–44). Such variations in A3G protein expression and IFN inducibility in liver cells may contribute to differences in re- sponse to treatment of HBV infections and different courses of viral pathogenesis in infected individuals.

There are several unique and novel contributions of this study, http://www.jimmunol.org/ ␥ FIGURE 5. IFN- induction of A3G is dependent on STAT1 in liver however, to both the understanding of A3G transcriptional regu- cells. A, Hep3B cells were treated with Jak inhibitor I (Jak) or DMSO for ␥ lation and the knowledge of IFN signaling pathways. First, we 1 h, and then treated with IFN- or control medium for 1 h. Equal amounts ␣ of cell lysates were run on separate SDS-PAGE gels to detect pSTAT1 demonstrated that transcriptional up-regulation of A3G by IFN- Tyr701 or total STAT1 proteins. B and C, Hep3B cells were treated with Jak was differentiated from other ISG and affected specifically by the inhibitor I (Jak Inh I) or DMSO for 1 h, and then IFN-␥ or control medium drug Rottlerin in Hep3B liver cells through a mechanism indepen- was added to the culture for 8 h. Induction of A3G or IRF-1 was measured dent of PKC-␦ and STAT1 activation (Fig. 3). Rottlerin has been by qRT-PCR. Error bars, SDs of fold inductions from triplicate cell sam- reported as a PKC-␦-specific inhibitor and used widely to impli- ples for each treatment. D and E, Hep3B cells transfected with siRNA and cate the role of PKC-␦ in various processes, including the tyrosine

␥ by guest on October 2, 2021 treated with IFN- or control medium as in Fig. 3A were processed for phosphorylation of STAT1 (35). However, one study found that ␥ qRT-PCR to measure induction of A3G and IRF-1 by IFN- . Error bars, Rottlerin potently inhibited multiple kinases in vitro, but had little SDs of fold inductions from triplicate cell samples. effect on PKC-␦ (45). It is possible that PKC-␦ may be inhibited by Rottlerin in vivo through an indirect mechanism (46), but our data suggest that PKC-␦ was not involved in the IFN-␣ induction of The functional ISRE is not within the proximal 135 bp 5Ј of the A3G. Further study will be required to identify the target of Rot- first exon of the A3G gene terlin responsible for the specific IFN regulation of A3G. ␣ It was published previously that the ISRE of the A3G gene was Second, we observed novel STAT1-independent IFN- induc- located with a 135-bp fragment from the 5Ј region before the first tion of A3G and other ISG in liver cells. The canonical models of exon of the A3G gene (24). We therefore wanted to confirm this IFN signaling pathways specify that the ISG factor 3 (ISGF3) result by cloning the identical fragment as published into the complex consisting of STAT1, STAT2, and IRF-9 is important for pGL2-basic luciferase reporter plasmid (pGL2-135). This frag- gene transcription in response to IFN-␣. In our study, we used ment is identical with that cloned into p127/ϩ7, as published in siRNA against these three transcripts in Hep3B cells to show that Tanaka et al. (24) (confirmed by sequencing). However, no basal only STAT2 and IRF-9, but not STAT1, were necessary for in- promoter activity nor IFN-␣-inducible activity was observed when duction of A3G and other genes by IFN-␣. It may be argued that we tested the construct in HepG2 cells as in the original study (Fig. complete knockdown of STAT1 was not achieved, and therefore 8). The positive control plasmid showed high promoter activity, residual proteins may be sufficient to mediate transcription. How- indicating that the cells were transfectable and the assay was func- ever, STAT1 shutdown was sufficient to inhibit the IFN-␥ pathway tional. Therefore, the functional ISRE is not within this 135-bp of gene transcription (Fig. 5) in liver cells. Furthermore, similar fragment and remains to be identified. levels of protein reduction were achieved with STAT1 and STAT2 siRNA (Fig. 4A), and STAT2 siRNA was able to inhibit the IFN- Discussion ␣-mediated induction of ISG (Fig. 4, B–F). Finally, the same A3G has potent antiviral activity against diverse retroviruses (3–8) siRNA strategy was used in 293T cells to block STAT1-dependent and HBV (21, 22), and can be classified among other well-known gene transcription in that cell line (Fig. 7). ISGs, such as PKR, ISG15, and MX1, which mediate a variety of At first glance, it may not be surprising that we found that antiviral effects. IFN induction of A3G is cell-type dependent, un- STAT2 and IRF-9 were required for induction of ISG in the like other ISG, such as PKR, which can be stimulated in all cell Hep3B cell line. It has been demonstrated that STAT2 can ho- types. A3G can be induced by IFN in liver cells, both primary modimerize and mediate gene transcription in conjunction with hepatocytes and liver cell lines, and primary macrophages, but not IRF-9 in the absence of STAT1. However, it was argued that the in primary CD4ϩ T cells or H9 cells. Although it may be argued DNA-binding affinity of such a complex was weak (36) and The Journal of Immunology 4537 Downloaded from http://www.jimmunol.org/

FIGURE 6. IRF-9 is required for IFN-␣ induction of A3G and other ISG in Hep3B cells. A, Hep3B cells were transfected with control, IRF-9, or IRF-9 plus STAT1, or IRF-9 plus STAT2 siRNA for 4 days, then treated with IFN-␣, IFN-␥, or control medium for 8 h. IRF-9 and ribosome p19 (loading control) proteins were detected by immunoblotting. B and C, Protein samples from cells transfected in A were run on separate gels, and STAT1 or STAT2 and by guest on October 2, 2021 ribosome p19 proteins were detected by immunoblotting. D–H, Hep3B cells treated with siRNA as in A were processed for qRT-PCR to measure induction of A3G, PKR, LMP2, ISG15, or MX1 by IFN-␣ in siRNA-treated cells over cells treated with control medium. Error bars, SEs of fold inductions from duplicate samples.

STAT1 provided additional stability to increase the trans activa- address whether IFN-␣ may also up-regulate ISG in primary hepa- tional function of the trimeric ISGF3 complex. That study was tocytes via a STAT1-independent pathway as well. Although performed in STAT1-negative cells (U3A) derived from the pa- STAT-independent IFN-␥ pathways of transcriptional activation rental 2fGTH epithelial cells. In fact, we have tested induction of are known (47, 48), to our knowledge this is the first report of an A3G and other genes by IFN-␣ and IFN-␥ in 2fTGH, U3A, and IFN-␣-independent pathway of up-regulating antiviral genes. U6A (STAT2-negative) cells. A3G was only minimally induced to Because signaling through Jak (Figs. 4, H and I, and 5, B and C), ϳ3-fold in the parental cells and not induced in the STAT1- and STAT2 (Fig. 4), and IRF-9 (Fig. 6) is essential for ISG induction STAT2-negative cells (data not shown). PKR was not induced in in these cells, our study opens questions as to which additional either the STAT1-negative or STAT2-negative cells by IFN-␣, and molecules may mediate this pathway. There are seven members of IRF-1 was not induced in the STAT1-negative cells by IFN-␥,as the STAT family of proteins that become activated by dimerization expected from the classical models (data not shown). We had ob- through Src homology 2 interactions upon tyrosine phosphoryla- tained similar results in 293T cells treated with STAT1 or STAT2 tion by Jak. Most of the STAT proteins can homodimerize, and it siRNA (Fig. 6). is possible that STAT2 may be acting as a homodimer in complex The unique observation in the present study, however, was that with IRF-9 to mediate transcription in the liver cells. However, reduction of STAT1 by siRNA did not even partially reduce the STAT1 is known to heterodimerize with STAT2 and STAT3 (31) transcriptional activation of certain ISG by IFN-␣ in the liver in response to IFN. Furthermore, STAT3 is activated (phosphor- Hep3B cells (Fig. 4). Therefore, our results suggest that a fully ylated at Tyr705) in response to IFN-␣ in liver cells (49) as well as functional STAT1-independent IFN-␣ pathway is most likely macrophages and T cells (data not shown), although its role in functioning either in parallel with the classically described ISGF3 gene transcription is unknown. If the alternative pathway to the complex or in the absence of STAT1 (31). We believe that the classical STAT1:STAT2:IRF-9 complex were a STAT3:STAT2: STAT1 independence of gene induction by IFN-␣ is unique to IRF-9 complex, then we would observe an effect on IFN induction liver cells because the phenomenon of STAT1-independent tran- of ISG if both STAT1 and STAT3 were reduced by siRNA. How- scription was observed specifically in Hep3B cells (Fig. 4) and ever, we did not observe a role for STAT3 in induction of A3G or Huh7 cells (data not shown), but not in 293T cells (Fig. 6) or other ISG because their up-regulation was not affected by STAT3 2fTGH cells (data not shown). Future studies will be needed to siRNA or STAT1 plus STAT3 siRNA treatment (data not shown). 4538 STAT1-INDEPENDENT REGULATION OF A3G BY IFN-␣ Downloaded from http://www.jimmunol.org/

FIGURE 7. STAT1 is required for both IFN-␣ and IFN-␥ induction of ISG in 293T cells. A, 293T cells were transfected with control, STAT1, or STAT2 siRNA for 4 days, and then treated with IFN-␣, IFN-␥, or control medium for 8 h. STAT1, STAT2, and ribosome p19 (loading control) proteins were detected by immunoblotting. B–H, 293T cells treated as in A were processed for qRT-PCR to measure induction of A3G, ISG15, MX1, and LMP2 by IFN-␣ or IRF-1 and LMP2 by IFN-␥. Error bars, SDs of fold inductions from triplicate cell samples. by guest on October 2, 2021

Although primary hepatocytes from STAT1 genetic knockout the STAT1-independent transcriptional complex requires both mice have been shown to display enhanced STAT3 signaling STAT2 and IRF-9, which mediate binding to DNA, it may bind to (phosphorylation at Tyr705) in response to IFN-␥ (50), we did not the same ISRE as used by the ISGF3 complex of the classical observe enhanced STAT3 signaling by IFN-␣ in STAT1 siRNA- pathway. However, the promoter of the A3G gene remains to be treated Hep3B cells (data not shown). Therefore, STAT3 is most likely not involved in an alternative pathway of STAT1-indepen- dent IFN-␣-mediated transcription in liver cells. Because STAT2 and IRF-9 are required for A3G induction by IFN-␣, and STAT2 and IRF-9 have been shown to bind DNA without STAT1 (36), it is likely that this transcriptional complex binds to some version of an ISRE. The various STAT hetero/ho- modimers bind to an 8- to 10-bp inverted repeated element with a variable ISRE sequence with the consensus sequence of 5Ј- Ј TT(N4–6)AA-3 (51). A recent study by Tanaka et al. (24) had reported on the identification of the ISRE in the 5Ј region before the first exon of the A3G gene. However, we did not observe basal FIGURE 8. The functional ISRE is not within the proximal 135 bp 5Ј of promoter activity nor IFN-␣-inducible activity when the construct the first exon of the A3G gene. The 135-bp fragment from the 5Ј region was tested as in the original study (Fig. 8). We believe that the before the first exon of the A3G gene was cloned into the pGL2-basic elements responsible for the A3G gene up-regulation in response luciferase reporter plasmid (pGL2-135). This fragment is identical with to IFNs remain to be identified, as well as the core promoter region that cloned into p127/ϩ7, as published in Tanaka et al. (24). HepG2 cells providing basal promoter activity in the absence of IFN were cotransfected with pSV-␤-galactosidase and either the pGL2-basic stimulation. (no promoter), pGL2–135, or positive control plasmid (pGL2 control con- The classical Jak/STAT mechanisms of IFN transcription are taining SV40 promoter) for 24 h. The cells were then treated with or with- out IFN-␣ (1000 IU/ml) for 9 h. Luciferase activity was measured by using well described, but cell type-specific signaling pathways are not the Luciferase Assay System (Promega) and the Multilabel Counter clearly understood. We summarize our findings with a proposed (WALLAC1420), and normalized by measuring ␤-galactosidase activity ␣ model of STAT1-independent IFN- pathway of induction of A3G using the ␤-Galactosidase Assay System (Promega). The lucif- and other ISG in liver cells (Fig. 9). The alternative STAT1-inde- erase activity is shown as units relative to the activity from cells transfected pendent pathway may occur in parallel with the classical Jak/ with pGL2-basic (without IFN) set at 1 unit. Error bars, SD of triplicate STAT pathway or is activated in the absence of STAT1. Because samples. The Journal of Immunology 4539

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