Hepatitis C Virus NS2 Protein Inhibits Gene Expression from Different Cellular and Viral Promoters in Hepatic and Nonhepatic Cell Lines

Virology 305, 260–266 (2003) doi:10.1006/viro.2002.1701

Franz Ludwig Dumoulin,*,1 Annette von dem Bussche,† Jisu Li,† Leila Khamzina,† Jack R.Wands,† Tilman Sauerbruch,* and Ulrich Spengler*

*Department of Medicine I, University of Bonn, Sigmund Freud Strasse 25, D-53127 Bonn, Germany; and †Liver Research Center, Rhode Island Hospital and Brown University School of Medicine, 55 Claverick Street, Providence, Rhode Island 02903 Received May 6, 2002; returned to author for revision June 18, 2002; accepted August 5, 2002

The mechanisms leadingto viral persistence and hepatocarcinogenesisin hepatitis C virus (HCV) infection are not fully understood. Recently, evidence has been accumulated that HCV viral proteins might interfere with gene expression of host cells. Here, we demonstrate that the amino-terminal portion of HCV NS2 protein inhibits the expression of reporter genes driven by different promoters or enhancer elements and also inhibits hepatitis B virus (HBV) gene expression and HBV DNA replication. The inhibitory effect of HCV NS2 on liver and non-liver-specific promoters and enhancer elements might be relevant for the pathogenesis of chronic HCV infection. © 2003 Elsevier Science (USA)

INTRODUCTION that NS2 is a transmembrane protein (Grakoui et al., 1993; Santolini et al., 1995).NS2 proteolytic activity is Hepatitis C virus (HCV) infection leads to chronic liver abolished by mutations of amino acid residues 952 and disease with the possible consequences of cirrhosis and 993 of the molecule (Grakoui et al., 1993; Hijikata et al., hepatocellular carcinoma (Bartenschlager and Lohmann, 1993) and the catalytic activity is associated with the 2000; De Francesco, 1999; Heintges and Wands, 1997; carboxyl-terminal part of the molecule (Pallaoro et al., Shimotohno, 1995).The mechanisms leading to viral per- 2001; Thibeault et al., 2001).In addition, it has been sistence and hepatocarcinogenesis are not fully under- shown that NS2 might also be important for the phos- stood.Recent evidence suggests that HCV viral proteins phorylation of NS5A (Liu et al., 1999).To date, an inter- could interfere with the gene expression of host cells.For action of NS2 protein with host cells has not been de- example, HCV core protein was shown to modify tran- scribed.We have therefore investigated possible inter- scription from a variety of promoter structures (Na- actions of NS2 with different hepatic and nonhepatic cell ganuma et al., 2000; Ray et al., 1995, 1997, 1998), to alter lines using reporter gene and in vitro HBV replication the activity of Rous sarcoma virus long terminal repeat assays. (LTR), simian virus (SV) 40 early promoters, and human immunodeficiency virus type 1 (HIV-1) LTR and to inhibit RESULTS AND DISCUSSION hepatitis B virus (HBV) and HIV replication (Ray et al., 1997; Srinivas et al., 1996; Shih et al., 1993).Moreover, it We first investigated the effects of HCV NS2 on differ- could be demonstrated that HCV NS4B protein activates ent cellular and viral promoters in human hepatic and reporter constructs containing a variety of inducible cis- nonhepatic cell lines using cotransfection experiments enhancer elements (Kato et al., 2000) and that HCV NS5A with luciferase reporter constructs under the control of protein acts as a potent transcriptional activator and as cytomegalovirus (CMV) immediate-early promoter.The a modulator of cell-cycle regulatory genes and cell expression of NS2 full-length and amino-terminal trun- growth (Chung et al., 1997; Ghosh et al., 1999; Kato et al., cated constructs was determined by Western blot (Fig. 1997). 1).Cotransfection of HepG2 cells with NS2 expression HCV NS2 protein is the first nonstructural protein en- plasmids resulted in a specific and highly reproducible coded by the HCV genome and possesses the function 70–80% reduction in luciferase activity which was not of an NS2/NS3 auto-protease (De Francesco, 1999; Ned- observed after cotransfection with HCV plasmids encod- dermann et al., 1997).The function depends on the pres- ing Core, E1, or p7, respectively (Fig.2A).The inhibitory ence of microsomal membranes and there is evidence effect of HCV NS2 on luciferase expression was dose- dependent (Fig.2B).Moreover, cotransfection with the NS2 expression vector resulted—with some variabil- 1 To whom correspondence and reprint requests should be ad- ity—in significantly decreased luciferase activity in the dressed.Fax: ϩϩ49/228/287-4323.E-mail: [email protected]. human hepatoma cell line Huh7 as well as in the non-

48 h after transient transfection with NS2 expression constructs (Fig.4). Considering the broad inhibitory effect of NS2 on different reporter gene constructs, we further investigated whether HCV NS2 sequences might also interfere with HBV gene expression and replication.For these experiments, Huh7 cells were cotransfected with HBV DNA (dimer) (Tong et al., 1990) and different HCV NS2 or control constructs.Interestingly, expression of full-length NS2 significantly reduced the secretion of HBV surface antigen (HBsAg) and e antigen (HBeAg) in the culture supernatant.HBV DNA replication, which is dependent on transcription of pregenomic RNA by the core promoter, was also moderately inhibited (Figs.5A and 5B). Finally, to better understand the structural basis of FIG. 1. Expression of HCV NS 2 sequences in human hepatoma cells. Human Huh7 hepatoma cells were transfected with different expres- NS2-mediated inhibition of gene expression and to rule sion vectors and cell lysates were prepared 48 h posttransfection for out unwanted effects due to the expression of NS2 as Western blots.NS2 expression was detected with rabbit polyclonal single viral protein, several expression constructs con- antiserum against NS2.Lane 1: pCMVLIC-NS2/amino acids (AA) 827- taining different parts of HCV NS2 in the molecular con- 1026/STOP 828; Lane 2: empty vector pCMVLIC; Lane 3: GFP transfec- text of adjacent regions (E2-p7) were cloned.Again, tion control construct (pGreenlantern; Life Technologies; Eggenstein, Germany); Lane 4: pCMVLIC-NS2 AA 810-1026 (full-length NS2); Lane 5 these plasmids were cotransfected with the pCMVLIC- pCMVLIC-NS2/AA827–1026 (N-terminal truncation). LUC reporter plasmid into the hepatic cell lines HepG2 or Huh7.While all constructs containing the amino-terminal portion of the HCV NS2 sequence (amino acids hepatic cell lines HeLa (cervix carcinoma), Jurkat (lym- 810–940) resulted in a significant decrease in luciferase phoma), and NIH 3T3 (murine fibroblasts; Fig.2C). activity, this effect was not observed for constructs con- We next investigated the influence of HCV NS2 on taining sequences from the carboxyl-terminal part of the other cellular and viral promoters.In these experiments, NS2 (Fig.6). reporter constructs encoding luciferase under the control Taken together, the data suggest a broad inhibitory of different promoter/enhancer structures were cotrans- effect of HCV NS2 protein.The effect could be de- fected with the full-length NS2 construct (pCMVLIC- tected—with some variability—in cell lines of different NS2/AA 810-1026) or empty vector control.As shown in origin.This variability might be due to the differences in Fig.2D, NS2 inhibited luciferase expression under the transfection efficiency.Alternatively, the variability might control of the human full-length tumor necrosis factor reflect a cell-specific susceptibility for the inhibitory effect of NS2.Moreover, the inhibitory effect was observed (TNF) alpha promoter and a minimal TNF alpha promoter for a variety of cellular and viral promoters, including lacking NFkappaB sites, as well as under control of inhibition of in vitro HBV replication and gene expression heterologous SV40 promoter/enhancer sequences, from HBV liver-specific (core) and non-liver-specific (S) NFkappaB binding sites, and the human ferrochelatase promoters.Thus, the effect could be detected using promoter, which is devoid of general cis-elements such structurally different promoter or enhancer structures, as TATA or CAAT boxes (Magness et al., 1998). including heterologous (CMV immediate early, SV-40) To rule out cytotoxic effects, HeLa tet-off cells were and “conventional” promoters (human TNF alpha) as well transfected with plasmid-encoding NS2 (or NS2 anti- as minimal promoters/response elements (minimal TNF sense as a control) and enhanced green fluorescent alpha promoter, NFkappa B binding sites) and promoter protein (EGFP) from a bidirectional, tetracycline-respon- structures devoid of general cis-elements such as TATA sive promoter.Expression of the construct was induced and CAAT boxes (ferrochelatase promoter).Several find- by withdrawal of tetracycline for 48 h after transfection ings demonstrate that this broad inhibitory effect of NS2 and the expression of NS2 and EGFP was analyzed by is nevertheless specific: First, inhibition was dose-de- indirect immunofluorescence or direct fluorescence mi- pendent and could be observed only with expression of croscopy, respectively (Fig.3).Transfection with a control NS2 but not with expression of other HCV structural vector showed a strong EGFP expression (Fig.3A).In proteins.Second, transient expression of NS2 did not contrast, EGFP fluorescence was suppressed to very low result in cytotoxic effects.Third, the effect could be levels (Fig.3B) after transfection with the bidirectional reproduced in different experimental systems such as HCV NS2/EGFP expression construct.In addition, we did reporter-gene assays or HBV replication in a permissive not observe any signs of cytotoxicity using lactate dehy- cell line.Fourth, the inhibition of reporter-gene expres- drogenase (LDH) assays for HepG2 or HeLa tet-off cells sion or HBV replication was also observed when NS2 262 DUMOULIN ET AL.

FIG. 2. 2A HCV NS2 inhibits CMV immediate early promoter.HepG2 cells were cotransfected with different HCV expression vectors and a CMV-driven luciferase reporter plasmid.Cotransfection with an HCV NS2 expression vector decreased luciferase activity by 70–80% relative to vect or control, while no significant reduction was observed after cotransfection with other expression constructs encoding for HCV core, E1, or p7 protein. (B) The inhibitory effect of HCV NS2 is dose-dependent.HepG2 cells were cotransfected with constant amounts of luciferase reporter construct and increasing amounts of NS2 expression plasmid adding empty vector to keep the total amount of transfected DNA constant in all transfections.(C) The inhibitory effect of HCV NS2 in non-hepatic cell lines.Huh 7 (human hepatoma), HeLa (human cervix carcinoma), Jurkat (human lymphoma), and NIH 3T3 cells (murine fibroblasts) were cotransfected with equal amounts of CMV-driven luciferase reporter plasmid and pCMVLIC-NS2/AA810–1026 (open bars) or empty vector control (filled bars).Cotransfection with the NS2 expression vector resulted in significantly decreased reporter gene activity with some variability in cell lines of different origin.(D) HCV NS2 inhibits multiple cellular and viral promoters/enhancers.The effect of HCV NS2 luciferase reporter-gene constructs under the control of different structural promoter or enhancer elements was assessed in HepG2 cells.HCV NS2 sequences inhibited reporter-gene expression to varying extents from human ferrochelatase promoter, an NFkappa B-luciferase construct, a full-length and a minimal human TNF alpha promoter without NFkappa B binding sites, and two viral promoters (SV-40 and CMV immediate early promoter).All data in this figure are given as mean Ϯ standard deviation of luciferase activity relative to vector control determined 48 h after transfection. protein was expressed in the context of adjacent pro- general transcription factor and/or translation (e.g., by teins.Fifth, the effect was specifically associated with protein–protein or protein–RNA/DNA interactions). expression of the amino-terminal part of NS2 protein. This could explain the relatively broad inhibitory effect The observed inhibition of cellular and viral promoters of NS2. represents a hitherto unknown activity of NS2 protein With regard to possible functional consequences, the due to a yet unknown mechanism.Mapping experiments inhibition of HBV in vitro replication and secretion of HVB narrowed down the inhibitory sequence to the amino- viral proteins by HCV NS2 is similar to the effect de- terminal portion (AA 810–940) of NS2 protein.Since the scribed for HCV core protein and could contribute to the NS2/NS3 auto-protease activity is mediated by the car- low HBV replication status observed in some patients boxyl-terminus of NS2 (Grakoui et al., 1993; Pallaoro et with HBV/HCV coinfection (Lee et al., 1997; Sagnelli et al., 2001; Thibeault et al., 2001), it is unlikely that the al., 2001).In addition, modification of cellular gene ex- inhibitory effect is caused by NS2 proteolytic activity. pression by HCV NS2 could importantly contribute to the An alternative mechanism might be the interference of pathogenesis of chronic hepatitis C and HCV-associated HCV NS2 protein with transcription, in particular with a hepatocarcinogenesis. HEPATITIS C VIRUS NS2 PROTEIN INHIBITS GENE EXPRESSION 263

FIG. 3. HCV NS2 inhibits EGFP expression from a bidirectional plasmid in HeLa tet-off cells.HeLa tet-off cells were transiently transfected with the bidirectional expression vector pBiEGFP-NS2/AA 810-1026 encoding full-length NS2 (B and D) and a control plasmid containing NS2 sequences in an antisense orientation (A and C).Expression from tetracycline-responsive promoter was induced by culture of the cells in the absence of tetracycline for 48 h.Cells were then processed for NS2-immunofluoresence and EGFP fluorescence.Transfection with antisense control showed bright EGFP fluorescence (A), while these cells displayed no specific NS2 immunoreactivity (C).In contrast, only background EGFP immunofluorescence could be detected in cells transfected with the NS2 full-length construct (B and D; magnification, ϫ400).

MATERIALS AND METHODS

Plasmids HCV-H strain sequences were PCR-amplified from a plasmid (Inchauspe et al., 1991) and cloned into the expression vectors pCMVLIC (Pharmingen, Hamburg, Germany), pcDNA 3.1(ϩ) (Invitrogen, San Diego, CA), and pBiEGFP (Clontech, Heidelberg, Germany) as previously described (Dumoulin et al., 1999).A Kozak consensus sequence was included for optimal translation efficiency (Kozak, 1986).The resulting vectors were pCMVLIC- Core/amino acids (AA) 1–191, -E1/AA 192–383, -p7/AA 747–809, -NS2/AA 810-1026.The pcDNA 3.1.-basedvec- tors were designated pcDNA3.1-NS2/AA 810-1026, -NS2- FIG. 4. Transient expression of HCV NS2 is not cytotoxic.HepG2 or delN/AA 827-1026, -NS2-STOP/AA 827-1026 with a pre- HeLa tet-off cells were transiently transfected with pCMVLIC-Core, pCMVLIC NS2/AA 810-1026, and vector control (HepG2) or pBiEGFP- mature stop codon at position 828.For expression of Core, pBiEGFP-NS2/AA 810-1026, or pBiEGFP-NS2 antisense control EGFP and NS2 from a bidirectional vector, HCV se- (HeLa tet-off) and expression from tetracycline-responsive promoter quences were cloned into pBi-EGFP either in sense or in was induced as described above.The regular 10% FCS medium was antisense orientation. replaced by 2% FCS medium 16 h after transfection and LDH activity was determined from the supernatant 48 h after transfection using a In addition, a panel of luciferase reporter constructs commercially available cytotoxicity detection kit (Roche Molecular Di- under control of different promoter/enhancers was used agnostics, Mannheim, Germany). in this study: human ferrochelatase promoter (pHFc-Luc; 264 DUMOULIN ET AL.

FIG. 5. HCV NS2 inhibits HBV DNA replication and production of HBsAg and HBeAg.Huh7 cells were cotransfected with HBV and either vector control, an HCV NS2 full-length expression vector, an HCV NS2 construct with a stop mutation immediately downstream the Kozak sequence, or an N-terminal truncated NS2 variant.HBV DNA replication was monitored at Day 5 after transfection (A); HBsAg and HBeAg secretion were detected by enzyme immunoassay or radioimmunoassay, respectively (B). a gift from David A.Brenner, University of North Carolina, 938-1141; GenBank Accession No.L11698; kindly pro- Chapel Hill, NC (Magness et al., 1998)), nuclear factor vided by Joachim Fandrey and Stilla Frede; University of (NF) kappaB (pNFkappaB-Luc containing 5ϫ NFkappaB Essen, Germany), simian virus 40 promoter/enhancer elements; Stratagene, Heidelberg, Germany), human tu- (pGL2; Promega, Heidelberg, Germany), and cytomega- mor necrosis factor (TNF)-alpha full-length promoter lovirus immediate early promoter (pCMVLIC-LUC; and human TNF-alpha promoter with a deletion of its Pharmingen).Finally, the following constructs encom- NFkappaB sites (nucleotides 1–1141 and nucleotides passing different parts of HCV NS2 and adjacent sequences were cloned: pCMVLIC-NS2/AA 940-1026, -NS2/AA 900-1026, -NS2/AA 810–940, -NS2/AA 810–900, E2-p7-NS2/AA 730-1006.

Cell lines and transfections HepG2 (human hepatoma), Jurkat (human CD4ϩ Lym- phoma), and HeLa (human cervix carcinoma) cells were purchased from DSMZ (Braunschweig, Germany); Huh 7 (human hepatoma) and NIH 3T3 (murine fibroblasts) cells were obtained from ATCC (Rockville, MD).HeLa tet-off cells stably expressing the tetracycline transacti- vator protein were acquired from Clontech.Cells were

cultured in a humidified 37°C/5% CO2 atmosphere in RPMI 1640 with glutamax (Life Technologies, Eggen- FIG. 6. The amino-terminal portion of HCV NS2 mediates the inhib- stein, Germany) supplemented with 10% fetal calf serum itory effect.Expression vectors were constructed encompassing differ- (Biochrom, Berlin, Germany), except for Huh 7 cells, ent parts of HCV NS2 and adjacent regions in pCMVLIC.Cotransfection which were cultured in minimal essential medium, 2 mM of HepG2 cells was carried out as described for Fig.2A.The inhibitory effect on luciferase activity was observed only after cotransfection with L-glutamine, nonessential amino acids, and 10% fetal calf sequences which included the amino-terminal portion of NS2 (amino serum (all chemicals from Sigma, St.Louis, MO).HepG2, acids 810-1026, 810–940, 730-1006). Jurkat, HeLa, and NIH3T3 cells were transfected for 16 h HEPATITIS C VIRUS NS2 PROTEIN INHIBITS GENE EXPRESSION 265 with DMRIE-C reagent (Life Technologies) at a 1:4 (␮g 48 h after transfection for LDH activity using the cytotox- DNA/␮l DMRIE-C) molar ratio; Huh7 cells were trans- icity detection kit (Roche Molecular Diagnostics, Mann- fected for 6 h using Mirus TransIT-LT1 (Panvera, Madi- heim, Germany).To avoid background LDH activity, cells son, WI) at a 1:3 (␮g DNA/␮l Mirus reagent) molar ratio. were grown in 2% FCS after transfection.LDH release All cell lines were grown in 24-well plates to 50–70% was determined from 100 ␮l supernatant and absor- confluency at the time of transfection; unless stated bance was measured at 480 nm using a reference wave- otherwise, 1:1 molar ratios of different plasmids were length at 620 nm. used for cotransfection assays. HBV DNA replication and production of HbsAgand Immunoblot HBeAg To verify expression from vector constructs, lysates Huh7 cells were cultured overnight to a density of were prepared from washed cells in hot Laemmli buffer 50–70% and cotransfected with HBV DNA (dimer) (Tong (Laemmli, 1970) 48 h after transfection.Lysates were et al., 1990) together with either vector control, HCV NS2 separated by 15% sodium dodecyl sulfate–polyacrylam- full-length expression vector (pcDNA3.1.-NS2/AA810- ide gel electrophoresis (SDS-PAGE) and blotted (Poly- 1026; NS2), HCV NS2 construct with a stop mutation screen membrane; NEN, Boston, MA).The blots were immediately downstream the Kozak sequence (pcDNA3.1.- blocked with 5% nonfat milk at room temperature for 30 NS2/AA827-1026-828TAG; NS2-Stop), or an N-terminal min and NS2 immunoreactivity was detected by chemi- truncated NS2 variant (pcDNA3.1.-NS2/AA940-1026; NS2- luminescence (SuperSignal West Pico; Pierce, Rockford, delN).HBV DNA replication was monitored at day 5 after IL) after overnight incubation with a 1:5000 dilution of transfection by Southern blot hybridization using HBV rabbit polyclonal antiserum against NS2 (WU 107, gener- DNA as a probe (Tong et al., 1990).HBsAg secretion was ously provided by Charles Rice, Rockefeller University, detected by enzyme immunoassay (Auszyme monoclo- New York, NY) (Grakoui et al., 1993; Fig.1). nal; Abbott Laboratories, Abbott Park, IL), HBeAg secretion by a radioimmunoassay (125I RIA Kit; DiaSorin, Still- Luciferase assays water, MN) according to the manufacturer’s instructions at 3 and 5 days after transfection. For luciferase assays, cells were lysed 48 h after cotransfection and luciferase activity was determined Statistics according to the manufacturer’s instructions (luciferase assay kit; Promega) using a Berthold Autolumat lumi- Statistical analysis using Student’s t tests was per- nometer (Berthold, Bergisch Gladbach, Germany).Lucif- formed with Microsoft Excel 97 (Microsoft Corp., Red- erase determinations were done in triplicate wells and mond, WA). results of cotransfection experiments are given as mean Ϯ standard deviation of luciferase activity relative ACKNOWLEDGMENT to vector control.At least three independent sets of The work was supported by grants from the Deutsche Gesellschaft experiments were performed with similar results. fu¨r Verdauungs- und Stoffwechselkrankheiten (Katsch Foundation Scholarship) and the Faculty of Medicine, University of Bonn, Germany Immunofluorescence (107/18 Bonfor).We are grateful to the following colleagues for provid- ing reagents: David A.Brenner, University of North Carolina, Chapel HeLa tet-off cells were grown on a coverslip, tran- Hill, North Carolina (pHFc-Luc); Joachim Fandrey and Stilla Frede, siently transfected with pBi-EGFP-NS2/AA 810-1026 or a University of Essen, Germany (TNF-alpha promoter reporter constructs); Genevie´ve Inchauspe´, Institut National de la Sante´etdela control construct containing NS2 sequence in an anti- Recherche Me´dicale, Lyon, France (HCV-H plasmid); and Charles Rice, sense orientation and fixed for 20 min at room temper- Rockefeller University, New York, NY (NS2 antiserum WU170).We also ature in 4% paraformaldehyde (Sigma, Deisenhofen, Ger- thank Darius Moradpour (University of Freiburg, Germany) for helpful many).Cells were permeabilized for 10 min at room discussions and Jennifer So¨hne for technical assistance.This work has temperature with 0.01% Triton X-100 in phosphate-buff- been presented at the Annual Meeting of the American Gastroenter- ological Association, San Francisco, CA, May 2002 (Gastroenterology ered saline (Sigma) and incubated overnight at 4°C with 2002, 122, 160). the rabbit polyclonal NS2 antiserum WU-107 at a 1:500 dilution.Bound antibody was detected with rhodamine- REFERENCES labeled goat anti-rabbit antibody (Dako, Hamburg, Ger- many; 1:1000) and viewed with a Leitz fluorescence mi- Bartenschlager, R.,and Lohmann, V.(2000).Replication of hepatitis C virus. 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