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Journal of Virology, May 2009, p. 5137-5147, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02179-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein {triangledown}

Hiromichi Hara,1,2 Hideki Aizaki,1 Mami Matsuda,1 Fumiko Shinkai-Ouchi,3 Yasushi Inoue,1,4 Kyoko Murakami,1 Ikuo Shoji,1,5 Hayato Kawakami,6 Yoshiharu Matsuura,7 Michael M. C. Lai,8 Tatsuo Miyamura,1 Takaji Wakita,1 and Tetsuro Suzuki1*

Department of Virology II,1 Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan,3 Department of Internal medicine, Division of Pulmonary Diseases, The Jikei University School of Medicine, Tokyo 105-8461, Japan,2 Mita Hospital, International University of Health and Welfare, Tokyo 108-8329, Japan,4 Division of Microbiology, Kobe University Graduate School of Medicine, Hyogo 650-0017, Japan,5 Department of Anatomy, Kyorin University School of Medicine, Tokyo 181-8611, Japan,6 Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan,7 Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, California 900338

Received 15 October 2008/ Accepted 20 February 2009


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ABSTRACT
 
Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.


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INTRODUCTION
 
Hepatitis C virus (HCV) infection represents a significant global healthcare burden, and current estimates suggest that a minimum of 3% of the world's population is chronically infected (4, 19). The virus is responsible for many cases of severe chronic liver diseases, including cirrhosis and hepatocellular carcinoma (4, 16, 19). HCV is a positive-stranded RNA virus belonging to the family Flaviviridae. Its ~9.6-kb genome is translated into a single polypeptide of about 3,000 amino acids (aa), in which the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B reside in the C-terminal half region (6, 34, 44). NS4A, a small 7-kDa protein, functions as a cofactor for NS3 to enhance NS3 enzyme activities such as serine protease and helicase activities. The hydrophobic N-terminal region of NS4A, which is predicted to form a transmembrane {alpha}-helix, is responsible for membrane anchorage of the NS3-4A complex (8, 44, 50), and the central region of NS4A is important for the interaction with NS3 (10, 44). A recent study demonstrated the involvement of the C terminus of NS4A in the regulation of NS5A hyperphosphorylation and viral replication (28).

The development of HCV replicon technology several years ago accelerated research on viral RNA replication (7, 44). Furthermore, a robust cell culture system for propagation of infectious HCV particles was developed using a viral genome of HCV genotype 2a, JFH-1 strain, enabling us to study every process in the viral life cycle (27, 47, 54). RNA derived from genotype 1a, HCV H77, containing cell-culture adaptive mutations, also produces infectious viruses (52). Using these systems, it has been reported that the HCV genome replicates in a distinct, subcellular replication complex (RC) compartment, which includes NS3-5B and the viral RNA (2, 14, 33). The RC forms in a distinct compartment with high concentrations of viral and cellular components located on detergent-resistant membrane (DRM) structures, possibly a lipid-raft structure (2, 41), which may protect the RC from external proteases and nucleases. Almost all processes in viral replication are dependent on the host cell's machinery and involve intimate interaction between viral and host proteins. However, the functional roles of host factors interacting with the HCV RC in viral genome replication remain ambiguous.

To gain a better understanding of cellular factors that are components of the HCV RC and that function as regulators of viral replication, a comparative proteomic analysis of DRM fractions from HCV replicon and parental cells and subsequent RNA interference (RNAi) silencing of selected genes were performed. We identified creatine kinase B (CKB) as a key factor for the HCV genome replication. CKB catalyzes the reversible transfer of the phosphate group of phosphocreatine (pCr) to ADP to yield ATP and creatine and is known to play important roles in local delivery and cellular compartmentalization of ATP (48, 51). The findings obtained here suggest that recruitment of CKB to the HCV RC, through CKB interaction with NS4A, is essential for maintenance or enhancement of viral replicase activity.


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MATERIALS AND METHODS
 
Cell lines, antibodies, and reagents. Human hepatoma cell line Huh-7.5.1 (54) was kindly provided by Francis V. Chisari. Cell lines carrying subgenomic replicon RNAs, namely, SGR-N (41) and SGR-JFH1 (23), were derived from the HCV-N (17) and JFH-1 strains (24), respectively. Mouse monoclonal antibodies (MAbs) against HCV NS3 (Chemicon, Temecula, CA), NS4A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), NS5A (Biodesign, Saco, ME), NS5B (2), FLAG (M2; Sigma-Aldrich, St. Louis, MO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Chemicon), and Flotillin-1 (BD Biosciences, San Jose, CA) and polyclonal antibodies (PAbs) against CKB (mouse [Abnova, Taipei, Taiwan], goat [Santa Cruz]), hemagglutinin (HA; Sigma-Aldrich), and FLAG (Sigma-Aldrich) were used. Cyclocreatine (Ccr; also known as 2-imino-1-imidazolidineacetic acid), pCr, and phosphopyruvic acid (pPy) were purchased from Sigma-Aldrich. Recombinant CKB and pyruvate kinase (PK) were obtained from Acris (Herfold, Germany) and Calbiochem (San Diego, CA), respectively.

Proteome analysis. RC-rich membrane fractions of cells were isolated as described previously (2, 41). Briefly, cells were lysed in hypotonic buffer. After removing the nuclei, supernatants were treated with 1% NP-40 for 60 min, mixed with 70% sucrose, overlaid with 55 and 10% sucrose, and centrifuged at 38,000 rpm for 14 h. Proteins from membrane fractions were purified by using a 2D Clean-Up kit (GE Healthcare, Tokyo, Japan), followed by labeling with fluorescent dyes: Cy5 for replicon cells, Cy3 for parental cells, and Cy2 for the protein standard containing equal amounts of both cell samples. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was performed using Immobiline DryStrip as the first-dimension gel and 12.5% polyacrylamide gel as the second-dimension gel. The 2D-DIGE images were analyzed quantitatively using the DeCyder software (GE Healthcare). Student t test was performed on differences between the tested samples using DeCyder biological variation analysis module. Samples were analyzed in triplicate. The protein spots of interest were excised from the gel, subjected to in-gel digestion using trypsin or lysyl endopeptidase and analyzed by liquid chromatography (MAGIC 2002 System; Michrom Bioresources, Auburn, CA) directly connected to electrospray ionization-ion trap mass spectrometry (LCQ-decaXP; Thermo Electron Corp., Iwakura, Japan). The results were subjected to database (NCBInr) search by Mascot server software (Matrix Science, Boston, MA) for peptide assignment.

Plasmids. A human CKB cDNA (43; kindly provided by Oriental Yeast Corp., Tokyo, Japan) was inserted into the EcoRI site of pCAGGS, yielding pCAGCKB. To generate expression plasmids for HA-tagged versions of wild-type and deletion mutated CKB, the corresponding DNA fragments were amplified by PCR, followed by introduction into the BglII site of pCAGGS. A fragment representing the inactive mutant CKB-C283S was synthesized by PCR mutagenesis. To generate FLAG-tagged NS protein expression plasmids, DNA fragments encoding either NS3, NS4A, NS4B, NS5A, or NS5B protein were amplified from HCV strains NIHJ1 (1) and JFH-1 (23) by PCR, followed by cloning into the EcoRI-EcoRV sites of pcDNA3-MEF (20). To generate an HA-tagged NS3 expression plasmid, a fragment encoding NS3 with the HA tag sequence at its N terminus was inserted into pCAGGS.

siRNA transfection. The small interfering RNAs (siRNAs) targeted to CKB (CKB-1 [5'-UAAGACCUUCCUGGUGUGGTT-3'] and CKB-2 [5'-CGUCACCCUUGGUAGAGUUTT-3']) and the scramble negative control siRNA to CKB-2 (5'-GGCGUACUAGCUUAUUCGCTT-3') were purchased from Sigma. Cells in a 24-well plate were transfected with siRNA using HiPerFect transfection reagent (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. The siRNA sequences for the other genes used in the siRNA screening are available upon request.

HCV infection. Culture media from Huh-7 cells transfected with in vitro-transcribed RNA corresponding to the full-length JFH-1 (47) was collected, concentrated, and used for the infection assay (3).

Quantification of HCV core protein and RNA. To estimate the levels of HCV core protein, aliquots of culture supernatants or of cell lysates were assayed by using HCV Core enzyme-linked immunosorbent assay kits (5). Total RNA was isolated from harvested cells using TRIzol (Invitrogen, Carlsbad, CA). Copy numbers of the viral RNA were determined by reverse transcription-PCR (RT-PCR) (2, 36, 46).

Immunoprecipitation, immunoblot analysis, and immunofluorescence microscopy. The analyses, as well as DNA transfection, were performed essentially as previously described (42). Cells were lysed in immunoprecipitation lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM dithiothreitol, 1 mM calcium acetate). For immunoprecipitation, supernatants of cell lysates were precipitated with anti-FLAG antibody and protein A-Sepharose Fast Flow beads (GE healthcare). For immunofluorescence microscopy, anti-CKB goat PAb and anti-NS4A MAb as primary antibodies and Alexa Fluor 555-conjugated donkey anti-goat immunoglobulin G (Invitrogen) and Alexa Fluor 488-conjugated rabbit anti-mouse immunoglobulin G (Invitrogen) as secondary antibodies were used and observed under an LSM 510 confocal microscope (Carl Zeiss, Oberkochen, Germany).

Immunoelectron microscopy. Postembedding immunostaining using the colloidal gold-labeling method was performed as described previously (38). Cells were fixed in 4% paraformaldehyde-1% glutaraldehyde at 4°C for 1 h. After dehydration through a graded series of ethanol, cells were embedded in LR White (London Resin Company, London, United Kingdom) and sectioned. After blocking, section grids were incubated with a mixture of anti-NS4A and anti-CKB antibodies at 4°C overnight, followed by treatment with a mixture of 18-nm colloidal gold-conjugated donkey anti-mouse immunoglobulin G and 12-nm colloidal gold-conjugated donkey anti-goat immunoglobulin G antibodies (Jackson Immunoresearch, West Grove, PA) at 4°C overnight. The sections were stained with uranyl acetate and observed under a transmission electron microscope.

Measurement of CK activity and cellular ATP level. Cells were lysed with passive lysis buffer (Promega, Madison, WI), and CK activities were measured based on Oliver methods (40), in which the activity of converting creatine phosphate and ADP to creatine and ATP was measured. ATP levels in cell lysates were measured by using a CellTiter-Glo luminescent cell viability assay (Promega).

RNA replication assays in permeabilized replicon cells and in vitro. The RNA synthesis assay using permeabilized replicon cells was based on a previously described method (33). Briefly, SGR-JFH1 cells were treated with 5 µg of actinomycin D/ml for 2 h, followed by permeabilization with 50 µg of digitonin/ml for 5 min. The resulting mix was incubated with 500 µM concentrations of ATP, GTP, and CTP; 10 µCi of UTP ([{alpha}-32P]UTP); 50 µg of actinomycin D/ml; and 5 mM pCr with or without 20 U of CKB/ml for 4 h at 27°C. RNA was extracted by using TRIzol and analyzed by 1% formaldehyde agarose gel electrophoresis. The cell-free RNA replication assay was performed as described previously (2).

In vitro helicase assays. Helicase activity on double-stranded RNA (dsRNA) was investigated as described previously (11) with some modifications. The 5' end of the release strand was labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (Ambion). The dsRNA substrate was obtained by annealing the labeled RNA with a template strand RNA at a molar ratio of 1:1. The helicase assay mixture contained 5 nM dsRNA, helicase enzyme (80 nM NS3 or NS3-4A [kindly provided by R. De Francesco]), 6 mM ATP, in the presence or absence of 20 U of CKB/ml in an assay buffer (25 mM MOPS-NaOH [pH 7.0], 2.5 mM dithiothreitol, 100 µg of bovine serum albumin/ml, 3 mM MgCl2, 5 mM pCr, 2.5 U of RNase inhibitor/ml). After the helicase reaction, samples were electrophoresed in a native 8% polyacrylamide gel and autoradiographed.

To determine the effect of PK/pPy system on the helicase activity, PK and pPy were used instead of CKB and pCr. Helicase activity on dsDNA was measured based on homogeneous time-resolved fluorescence quenching using a Trupoint helicase assay kit (Perkin-Elmer, Waltham, MA) according to the manufacturer's instructions.

In vitro protease assay. In vitro HCV protease activity of NS3-4A or NS3 was analyzed by using a SensoLyteHCV protease assay kit (AnaSpec, San Jose, CA) according to the manufacturer's instructions.


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RESULTS
 
Identification of host factors involved in HCV RNA replication by comparative proteomic analysis of DRM fractions and RNAi silencing. To identify host proteins involved in the HCV RC, proteome profiles of the RC-rich membrane fraction in Huh-7 cells harboring subgenomic replicon RNA derived from genotype 1b, N isolate (SGR-N) were compared to those of parental cells by 2D-DIGE. We confirmed that the DRM fraction obtained from SGR-N cells is functionally active in a cell-free replication assay (Fig. 1A). Three independent proteome experiments were performed for a reliable analysis of protein expression. Approximately 1,300 spots were resolved in each gel, and 4 to 5% of the protein spots represented a >2-fold increase in the membrane fraction of replicon cells in each experiment (Fig. 1B). The protein spots that exhibited high reproducibility (an example shown in Fig. 1C) were excised, digested by trypsin or lysyl endopeptidase, and analyzed by mass spectrometry, which identified the corresponding proteins in 27 cases (Table 1). Among the proteins implicated in a variety of functional categories, 10 were involved in protein folding, mainly as chaperones, 7 were metabolic and biosynthesis enzymes including proteins for redox regulation or energy pathways, 3 were involved in cytoskeleton organization, and 3 proteins were related to cellular processes, mainly proteolysis pathways. The viral NS proteins identified as differentially expressed proteins in the analysis were not listed.


Figure 1
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  		FIG. 1. Comparative proteomic analysis of DRM fractions and RNAi silencing. (A) Preparation of functionally active RC fraction for proteome analysis. DRM fractions obtained from SGR-N cells and parental Huh-7 cells were analyzed by immunoblotting with anti-NS5A and anti-NS5B antibodies (upper panel) and by the cell-free RNA replication assay (lower panel). (B) Histogram representation of proteins detected in 2D-DIGE. Images were analyzed quantitatively by the DeCyder software. The left and right y axis, respectively, indicate the spot frequency and the maximum volume of each spot, given against the log volume ratio (x axis). (C) Comparison of 2D-DIGE maps of proteins from DRM fractions of SGR-N cells and Huh-7 cells. Enlarged 2D-DIGE gel images of regions containing protein spots of CKB (arrows) are shown. (D) Effects of siRNAs of genes selected from comparative proteome analysis on HCV RNA replication. SGR-N cells were transfected with siRNA specific to cathepsin D, CKB (siCKB-1), GRP58, GST, Hsp70 protein 4, GRP94, HMG-coenzyme A synthase, or Tat binding protein 7 or with nontargeting (NC) siRNA. At 48 h posttransfection, total RNA was isolated and HCV RNA levels were assessed by real-time RT-PCR. (E) Enrichment of CKB in the DRM of HCV replicon cells. Equal amounts of DRM fractions from SGR-N and parental Huh-7 cells, or whole-cell lysates from both cells were analyzed by immunoblotting with antibodies against CKB, flotilin-1 or GAPDH.


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  		TABLE 1. Selected proteins that reproducibly increased in the DRM fraction of SGR-N cellsa

In order to identify host factors involved in HCV replication, we examined the effects on viral RNA replication of transfection of SGR-N cells with siRNAs against genes encoding nine proteins belonging to diverse classes of biological functions (Table 1). Each siRNA reduced the HCV RNA level to 47 to 76% of the level of the siRNA control (Fig. 1D). None of the siRNAs tested exhibited considerable cytotoxicity against the replicon cells, ruling out overt toxicity as a mechanism for inhibition of viral RNA replication. Among the candidate genes examined, we observed a reproducible inhibition of HCV RNA replication by two independent siRNAs targeting CKB (see below).

CKB participates in HCV RNA replication and the propagation of infectious virus. CKB is a brain-type creatine kinase isoenzyme and is also detected in a variety of other tissues, including human liver (32). Steady-state levels of CKB in the DRM fraction, as well as in whole-cell lysate of SGR-N cells were compared to those from parental cells by Western blotting. The CKB level in the DRM fraction of replicon cells was higher than that in parental cells (Fig. 1E), confirming the results of the proteome analysis described above. In contrast, the CKB level in whole cells was similar in both cells (Fig. 1E). These results suggest participation of posttranslational modification, such as translocation to the DRM fraction, of CKB in replicon cells.

Figure 2A shows the inhibitory effect on HCV RNA replication of CKB siRNA; siCKB-2, the sequence of which does not overlap with the sequence of siCKB-1 used in the above siRNA screening (Fig. 1D). Transfection with siCKB-2 effectively decreased the cellular level of CKB enzymatic activity (data not shown), as well as the abundance of CKB protein (Fig. 2A), and resulted in 60% reduction in the viral RNA level in SGR-N cells compared to the cells treated with control siRNA. This inhibitory effect of siRNA on HCV RNA abundance was also observed in JFH-1-derived subgenomic replicon (SGR-JFH1) cells. The viral RNA level in the cells transfected with siCKB-2 decreased by 50% compared to the control (Fig. 2A). We also tested the CKB mutant, CKB-C283S, in which Cys at aa 283, near the catalytic site, has been replaced with Ser (Fig. 3A) and which is known to be enzymatically inactive and to work in a dominant-negative manner (22, 29). As expected, overexpression of CKB-C283S resulted in a reduction in HCV RNA replication in SGR-N cells (Fig. 2B). We obtained a similar result in SGR-JFH1 cells, as described below (Fig. 3E).


Figure 2
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  		FIG. 2. Involvement of CKB in HCV replication. (A and E) Knockdown of endogenous CKB in SGR-N and SGR-JFH1 cells (A) or HCVcc-infected cells (E). Cells were transfected with siRNA against CKB (siCKB-2) or control siRNA (siCont) and were harvested at 72 h posttransfection. Real-time RT-PCR for HCV RNA levels and immunoblotting for CKB and GAPDH were performed. (B) SGR-N cells were transfected with pCAGCKB-C283S or empty vector, and HCV RNA levels and expression of CKB and CKB-C283S were determined 72 h posttransfection. SGR-N and SGR-JFH1 cells (C) or HCVcc-infected cells (F) were treated with Ccr at various concentrations for 72 h, followed by quantification of HCV RNAs and total cellular proteins. ATP levels (D) were determined after transfection with siCKB-2, pCAGCKB-C283S, or treatment with Ccr for 72 h in SGR-N cells. The ATP levels in the cells transfected with negative control siRNA (left), empty vector (middle), and no treatment (right) were set at 100%, respectively. (F) HCVcc-infected cells were treated with Ccr, and the viral core protein levels in cells (left) and supernatants (middle) were determined at 72 h postinfection. Collected culture supernatants were inoculated into naive Huh-7.5.1 cells after the removal of Ccr. After 72 h, the core proteins in cells were determined (right panel). All data are presented as averages and standard deviation values for at least triplicate samples. *, P < 0.05 against control such as transfection with siCont (A and E) or empty vector (B) or nontreatment (C, D, and F).


Figure 3
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  		FIG. 3. CKB interacts with HCV NS4A. (A) Structures of CKB constructs used in the present study. A full-length wild-type CKB without an epitope tag (CKB) or with an N-terminal HA tag (HA-CKB), HA-CKB with deletions (aa 1 to 357, aa 1 to 296, aa 1 to 247, aa 1 to 184, and aa 297 to 381 and del297-357), CKB mutant at the catalytic site, Cys-283 (CKB-C283S) or CKB-C283S lacking aa 297 to 357 (CKB-C283Sdel297-357) are shown. HA-CKB was coexpressed with FLAG-tagged versions of each NS protein of strain NIHJ1 (B) or with NS4A of strain JFH-1 (C) in 293T cells and immunoprecipitated (IP) with an anti-FLAG antibody. Immunoprecipitates were subjected to immunoblotting (IB) with anti-HA or anti-FLAG antibody. (D) Each CKB deletion mutant was coexpressed with FLAG-NS4A in 293T cells. Immunoprecipitates were analyzed by immunoblotting. Arrow, CKB; arrowhead, immunoglobulin heavy chain. (E) SGR-JFH1 cells were transfected with the expression plasmid for CKB-C283S, CKB-C283Sdel297-357 or empty vector. At 72 h posttransfection, HCV RNA levels and the expression of CKB and CKB-C283S were determined by real-time RT-PCR and immunoblotting with anti-HA antibody, respectively. For HCV RNA quantitation, data are indicated as averages and standard deviations (n = 3). *, P < 0.05 against the empty vector control. (F) Structure of NS4A and NS4A constructs. FLAG-tagged NS4A (aa 1 to 54) or its truncated mutants (aa 1 to 20, aa 21 to 39, or aa 40 to 54) are shown. (G) Each NS4A deletion mutant was coexpressed with HA-CKB and analyzed as described above. (H) FLAG-NS4A was coexpressed with HA-NS3 or HA-NS3 and CKB, followed by immunoprecipitation with anti-FLAG antibody. Immunoprecipitates were analyzed by immunoblotting with anti-HA, anti-FLAG or anti-CKB antibody.

To further examine the involvement of CKB in HCV RNA replication, we tested the effect of Ccr, a substrate analogue and possible inhibitor for CK in either SGR-N, SGR-JFH1 (Fig. 2C), or Huh7 cells transiently replicating luciferase-subgenomic replicon (data not shown). We found dose-dependent inhibition of HCV RNA replication but no observed effect on total cellular levels of protein and ATP (Fig. 2D) in the replicon setting used.

We next examined whether the knockdown of CKB or treatment with Ccr would abrogate the production of HCVcc. At 72 h posttransfection with siCKB-2, the HCV core level in cells infected with HCVcc was significantly reduced (Fig. 2E). Treatment of the infected cells with Ccr at various concentrations also reduced the intracellular and supernatant core level and subsequently decreased HCVcc production (Fig. 2F). These results demonstrate that suppression of the HCV RNA replication by the siRNA-mediated knockdown of CKB or treatment with CKB inhibitor leads to reduction of the production of infectious virus.

CKB interacts with HCV NS4A. Having established a role for CKB in HCV RNA replication, we then tried to determine to how CKB influences the HCV life cycle. It has been reported that interaction of CKB with some cellular proteins is required for local availability of CKB activity and local generation of ATP (22, 29). To examine the possible interaction of CKB with HCV NS proteins, HA-tagged CKB (HA-CKB) was coexpressed with FLAG-tagged NS proteins (NIHJ1 strain), followed by immunoprecipitation with an anti-FLAG antibody. CKB was shown to specifically interact with NS4A. No or little interaction was observed between CKB and either NS3, NS4B, NS5A, or NS5B (Fig. 3B). CKB-NS4A interaction was also found with the JFH-1 strain (Fig. 3C).

To identify the CKB region required for the interaction with NS4A, various deletion mutants of CKB were generated (Fig. 3A). An immunoprecipitation assay indicated that NS4A was coimmunoprecipitated with either a full-length CKB, a C-terminal deletion (aa 1 to 357), an N-terminal deletion (aa 297 to 381), or CKB-C283S, but not with aa 1 to 296, aa 1 to 247, or aa 1 to 184 (Fig. 3D, upper middle panel). Further, internal deletions of CKB (CKBdel297-357 and CKB-C283Sdel297-357) failed to interact with NS4A (Fig. 3D, lower panel), suggesting that aa 297 to 357 of CKB are important for its interaction with NS4A. It is noted that the expression of CKB aa 297 to 357 in cells was undetected, presumably due to its misfolding and/or instability. To verify a role for CKB-NS4A interaction in HCV RNA replication, we further determined the effect of expression of either CKB-C283S or its internal deletion lacking aa 297 to 357 (CKB-C283Sdel297-357) on viral replication in SGR-JFH1 cells. As expected, the HCV RNA level was significantly decreased by CKB-C283S, whereas this effect was not observed by CKB-C283Sdel297-357 (Fig. 3E).

NS4A is a 54-residue small protein composed of three domains: the N-terminal membrane anchor, the central domain responsible for interacting with NS3, and the C-terminal acidic domain. To define the portion in NS4A responsible for its interaction with CKB, we constructed three NS4A deletion mutants, each separately expressing one of the NS4A domains, with a FLAG tag (Fig. 3F). CKB proved to interact with the central domain, aa 21 to 39, of NS4A, which is involved in formation of the NS3-NS4A complex (Fig. 3G). We therefore investigate whether NS3-NS4A interaction is affected in the presence of CKB and found that exogenous expression of CKB has no influence on NS3-NS4A interaction, and a putative NS3-NS4A-CKB complex was detected in the coimmunoprecipitation analysis (Fig. 3H). Collectively, these results strongly suggest that CKB plays a key role in HCV RNA replication via interaction with NS4A.

Subcellular localization of CKB and NS4A in cells replicating HCV RNA. CKB is distributed throughout cells but is mainly localized in the perinuclear area (31), whereas NS4A is predominantly localized at the endoplasmic reticulum and mitochondrial membranes (37). We examined the possible subcellular colocalization of CKB and NS4A in SGR-N cells by immunofluorescence staining (Fig. 4A). CKB tended to gather in the perinuclear area of HCV replicating cells and was partially colocalized with NS4A in the area, sharing a dotlike structure. To further analyze the subcellular compartments in which CKB and NS4A coexist, we used double-labeling immunoelectron microscopy on SGR-N cells using antibodies against CKB and NS4A, with secondary antibodies coupled to 12- and 18-nm gold particles, respectively. One fraction of CKB colocalized with NS4A in the cytoplasmic electron-dense regions, presumably derived from altered or folded membrane structures (Fig. 4B, left panel) and mitochondria (Fig. 4B, right panel).


Figure 4
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  		FIG. 4. Colocalization of CKB with HCV NS4A. (A) Indirect immunofluorescence analysis. The primary antibodies used were anti-CKB goat PAb (red) and anti-NS4A MAb (green). Merged images of red and green signals are shown. High-magnification panels are enlarged images of white squares in the merge panels. (B) Immunoelectron microscopic localization of CKB and NS4A. SGR-N cells were double-immunolabeled for CKB (12-nm gold particles; white arrows) and for NS4A (18-nm gold particle; gray arrows). Mi, mitochondria. Bars, 200 nm.

CKB enhances functional HCV replicase and NS3-4A helicase. NS4A is known to mediate membrane association of the NS3-4A complex and to function as a cofactor in NS3 enzyme activity. To understand the mechanism(s) underlying positive regulation of HCV RNA replication through CKB via its interaction with NS4A, we first investigated whether CKB modulates NS3-4A helicase activity. NS3-4A helicase is a member of the superfamily-2 DexH/D-box helicase, which unwinds RNA-RNA substrates in a 3'-to-5' direction. During RNA replication, the NS3-4A helicase is believed to translocate along the nucleic acid substrate by changing its protein conformation, utilizing the energy of ATP hydrolysis (9). We then tested the effect of CKB on RNA- or DNA-unwinding activity using purified recombinant full-length NS3 and NS3-4A complex (12). As shown in Fig. 5A (left middle panel), both NS3 and NS3-4A helicase activity unwound dsRNA substrate most efficiently when CKB, ATP, and pCr were added to the reaction mixture. The enhancing effect of CKB was observed in the presence of pCr but not in the absence of it, suggesting that catalytic activity of CKB is important for its effect on the HCV helicase activity. Similar results were obtained from the DNA helicase assay using dsDNA substrate (Fig. 5B). To address the specificity of the stimulation by the CKB/pCr system, effects of PK and pPy, which are also involved in the ATP generation, were determined (Fig. 5A, right panels). Exogenous PK and pPy at the same concentrations as those of CKB and pCr used in the study exhibited no effect on the HCV helicase activity.


Figure 5
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  		FIG. 5. CKB enhances NS3-4A helicase and HCV replicase activities. (A) In vitro RNA helicase activity of NS3-4A or NS3 was determined by detecting unwound single-strand RNA (ss) derived from the partially dsRNA substrate (ds). Band intensities corresponding to unwound products and those to dsRNA substrates were determined by ImageQuant 5.2 (Molecular Dynamics), and the ssRNA/dsRNA ratios were calculated. The results are representative of three similar experiments. (B) In vitro DNA helicase activity of NS3-4A or NS3 was analyzed by using a commercially available kit. The data represent averages and standard deviations (n = 3). *, P < 0.05 against the value without supplementation of CKB and ATP. (C) The in vitro HCV protease activity of NS3-4A or NS3 in the presence or absence of CKB was analyzed. Error bars represent standard deviations (n = 3). (D) Replicase activity in permeabilized replicon cells. The upper panel shows the activity for synthesis of HCV subgenomic RNA in the digitonin-permeabilized SGR-JFH1 cells with or without supplementation of CKB was measured. The middle panel shows results for SGR-JFH1 or Huh-7 cells that were transfected with siCKB-2 or siCont and permeabilized at 72 h posttransfection. The permeabilized cells with or without supplementation of CKB were subjected to the replicase assay. The lower panel shows the immunoblotting results for whole-cell lysates of siRNA-transfected cells.

The effect of CKB on NS3-4A serine protease activity, which is considered to be ATP-independent, was also assessed in an in vitro protease assay using the purified viral proteins as mentioned above (Fig. 5C). As expected, NS3-4A complex exhibited significantly higher activity than NS3 alone; however, CKB did not affect the protease activities of NS3 or NS3-4A.

Finally, we investigated loss and gain of function of CKB in HCV replicase activity, which requires high-energy phosphate, in the context of semi-intact replicon cells. Miyanari et al. (33) reported that the function of the active HCV RC can be monitored in permeabilized replicon cells treated with digitonin. Thus, permeabilized replicon cells in the presence or absence of exogenous CKB were incubated with [{alpha}-32P]UTP to detect newly synthesized RNA. As indicated in Fig. 5D, an ~8-kb band corresponding to HCV subgenomic RNA was most abundant in cells in the presence of exogenous CKB, ATP and pCr. The enhancing effect of CKB was observed in the presence but not in the absence of pCr, suggesting that catalytic activity of CKB is important for its effect on the replicase activity. As for the RNA helicase assay, exogenous PK and pPy did not enhance the replicase activity (data not shown). HCV replicase activity in permeabilized cells to which we had introduced siCKB-2 was diminished compared to that in siRNA control-treated cells. Interestingly, the replicase activity in the CKB-depleted cells was recovered by the addition of CKB. Thus, our findings suggest that CKB functions as a key regulator of HCV genome replication by controlling energy-dependent viral enzyme activities.


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DISCUSSION
 
Viral replication requires energy and macromolecule synthesis, and host cells provide the viruses with metabolic resources necessary for their efficient replication. Thus, it is highly likely that interaction of viruses with host cell metabolic pathways, including energy-generating systems, contributes to the virus growth cycle. In the regulation of HCV genome replication, the functions of the viral NS proteins that comprise the RC might be regulated by association in individual host cell factors. For example, hVAP-A and -B function as cofactors of modulating RC formation via interacting with NS5A and NS5B (13, 18). Cyclophilin B is involved in stimulating viral RNA binding activity via interacting with NS5B (49). FKBP8 (39) and hB-ind1 (45) play an important role in recruiting Hsp90 to RC via interacting with NS5A. However, the association of viral protein(s) with the cellular energy-generating system to directly regulate the activity of the RC has not been well understood.

In the present study, the accumulation of CKB, an ATP-generating enzyme, in the HCV RC-rich membrane fraction of viral replicating cells and its importance in replication of the HCV genome and production of infectious virions have been demonstrated. Enzymatic analyses with semi-intact replicon cells and purified NS3-4A protein revealed that CKB enhances the functional replicase and helicase of HCV. Its enhancing effect was observed in the presence of pCr but not in its absence, suggesting that the catalytic activity of CKB is important for enhancing the replicase and helicase activities. Moreover, we clearly detected a CKB-NS4A complex using anti-tag antibodies in cotransfection experiments, but the endogenous complex could not be immunoprecipitated from cells expressing only endogenous levels of CKB, probably because of the inefficiency of the available antibodies. Further, a deletion of the NS4A-interacting region within an inactive mutant of CKB (CKB-C283S) resulted in the loss of its dominant-negative effect on HCV replication.

Creatine kinase, an evolutionarily conserved enzyme, is known to be critical for the maintenance and regulation of cellular energy stores in tissues with high and rapidly changing energy demands (48). In mammals, three cytosolic and two mitochondrial isoforms of CK, which share certain conserved regions, are expressed (35). The brain-type CK, CKB, plays a major role in cellular energy metabolism of nonmuscle cells, reversibly catalyzing the ATP-dependent phosphorylation of creatine and, hence, providing an ATP buffering system in subcellular compartments of high and fluctuating energy demand (21, 29). CKB is overexpressed in a wide range of tumor tissues and tumor cell lines, including hepatocellular carcinoma (32), and is used as a prognostic marker of cancer.

Although CK and creatine phosphate have been supplemented to in vitro replicase assays of some RNA viruses (15, 33), understanding of CKB function in the virus life cycle has been limited. One study indicated that the CK substrate analog, Ccr, exhibits antiviral activity against several herpesviruses but not influenza viruses or vesicular stomatitis virus (26). We have demonstrated here that HCV genome replication is downregulated by either treatment with Ccr, siRNA-mediated knockdown of CKB, or the exogenous expression of CKB-C283S. Coimmunoprecipitation experiments revealed that the essential domain within NS4A for the interaction with CKB is the NS4A central domain, aa 21 to 39, which is also responsible for NS3-4A complex formation. However, the NS3-4A interaction was not impaired by overexpression of CKB, and CKB was found to be able to form a complex with NS3-4A (Fig. 3H). Since CKB does not directly interact with NS3 (Fig. 3A), it is likely that NS3-4A-CKB association occurs through two interactions of NS3-4A and NS4A-CKB. We examined whether the formation of the ternary complex affects HCV enzymatic activities, possibly through conformational changes in the viral proteins, and found that CKB has no influence on NS3-4A protease activity (Fig. 5C). With regard to helicase activity, the effect of CKB on RNA unwinding activity by NS3-4A was similar to the effect of NS3 alone in the presence of ATP (Fig. 5A). It is conceivable that interaction with CKB causes no or little global change in the NS3-4A conformation and does not affect the viral helicase and protease activities.

In general, translation initiation in eukaryotes includes an ATP-dependent process such as unwinding the secondary structure in the 5'-untranslated region to permit assembly of 48S ribosomal complexes. It was reported, however, that 48S complex formation on the HCV internal ribosome entry site (IRES) has no requirement for ATP hydrolysis (25). In fact, we found that Huh-7 cells with or without gene silencing of CKB exhibited the same level of HCV IRES activity by transfection with IRES-reporter constructs (data not shown).

Collectively, we conclude that CKB is targeted to the HCV RC through its interaction with NS4A and functions as a positive regulator for the viral replicase by providing ATP. It is likely that the catalytic activity of CKB that associates with the viral RC is important for enhancing the RNA replication. The role of CKB-NS4A interaction in the enhancing effect seems to be limited. Although either knocking down CKB, expression of the dominant-negative mutant of CKB, or Ccr treatment resulted in the reduction of HCV replication (Fig. 2A to C), the total cellular ATP levels were not changed under these conditions (Fig. 2D). This suggests that CKB contributes to enhancing HCV replication through controlling the ATP level in the particular RC compartment. A tight coupling of a fast ATP regeneration and delivery system to the viral RC is advantageous for achieving efficient replication of the viral genome. To our knowledge, the findings presented here provide the first experimental evidence of the involvement of viral protein in recruiting an ATP generating/buffering system to the subcellular compartment for viral genome replication, a site with high-energy turnover. Given that the levels of HCV RNA were not dramatically diminished by the knocking down, dominant-negative mutant or Ccr, CKB may not be absolutely critical for the viral replication. One would argue that energy required for HCV genome replication can be partly complemented from the intracellular ATP pool.

Although there are several isoforms of CK as described above, the most abundant CK species expressed in Huh-7 cells in the present study was CKB, and no other isoenzymes, including mitochondrial CK, were detected by an isoform analysis based on the overlay gel technique (32; data not shown). Thus, the CKB isoenzyme appears to be a key molecule in the energy metabolism of HCV replicating cells. To identify potential HCV RC components, we used a comparative proteome analysis of the DRM fraction in cells harboring HCV subgenomic replicon and the DRM fractions in parental cells and then identified proteins that were more abundant in the fraction of HCV replicating cells. In agreement with similar previously reported approaches using the DRM or lipid raft fraction (30, 53), the functional categories of identified proteins included protein folding or assembly, cell metabolism and biosynthesis, cellular processes, and cytoskeleton organization (Table 1). Interestingly, Mannova et al. found that CKB was upregulated in the fraction of Huh-7 cells carrying the genotype 1b Con1 isolate-derived HCV replicon, as determined using stable isotope labeling by amino acids combined with one-dimensional electrophoresis (30). However, the effect of CKB on regulation of the HCV life cycle was not examined in that study.

In conclusion, CKB interacts with HCV NS4A and is important for efficient replication of the viral genome. Recruitment of CKB to the HCV replication machinery through its interaction with NS4A may have important implications for the maintenance or enhancement of the functional replicase activity in the RC compartment, where high-energy phosphoryl groups are required. A strategy for specific interception of energy supply at the subcellular site of HCV genome replication by disruption of the NS4A-CKB interface may lead to development of a new type of antiviral agent.


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ACKNOWLEDGMENTS
 
We thank Francis V. Chisari (The Scripps Research Institute) for providing Huh-7.5.1 cells; Raffaele De Francesco (Istituto di Ricerche di Biologia Molecolare, P. Angeletti) for providing purified recombinant NS3 and NS3-4A proteins; Oriental Yeast Co., Ltd., for providing human CKB cDNA; Minoru Fukuda (Laboratory for Electron Microscopy, Kyorin University School of Medicine) for electron microscopy; S. Yoshizaki, T. Shimoji, M. Kaga, M. Sasaki, and T. Date for technical assistance; and T. Mizoguchi for secretarial work.

This study was supported by a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science, from the Ministry of Health, Labor, and Welfare of Japan and from the Ministry of Education, Culture, Sports, Science, and Technology and by Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation, Japan, and by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111. Fax: 81-3-5285-1161. E-mail: tesuzuki{at}nih.go.jp Back

{triangledown} Published ahead of print on 4 March 2009. Back


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Journal of Virology, May 2009, p. 5137-5147, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02179-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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