Yasuhiro Itsui and Naoya Sakamoto contributed to this work.
Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein†
Article first published online: 5 AUG 2009
Copyright © 2009 American Association for the Study of Liver Diseases
Volume 50, Issue 6, pages 1727–1737, December 2009
How to Cite
Itsui, Y., Sakamoto, N., Kakinuma, S., Nakagawa, M., Sekine-Osajima, Y., Tasaka-Fujita, M., Nishimura-Sakurai, Y., Suda, G., Karakama, Y., Mishima, K., Yamamoto, M., Watanabe, T., Ueyama, M., Funaoka, Y., Azuma, S. and Watanabe, M. (2009), Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein. Hepatology, 50: 1727–1737. doi: 10.1002/hep.23195
Potential conflict of interest: Nothing to report.
- Issue published online: 20 NOV 2009
- Article first published online: 5 AUG 2009
- Accepted manuscript online: 5 AUG 2009 12:00AM EST
- Manuscript Accepted: 24 JUL 2009
- Manuscript Received: 12 MAR 2009
- Ministry of Education, Culture, Sports, Science and Technology of Japan
- Japan Society for the Promotion of Science
- Ministry of Health, Labour and Welfare
- Japan Health Sciences Foundation
- National Institute of Biomedical Innovation
- Top of page
- Materials and Methods
- Supporting Information
Interferons (IFNs) and the interferon-stimulated genes (ISGs) play a central role in antiviral responses against hepatitis C virus (HCV) infection. We have reported previously that ISGs, including guanylate binding protein 1 (GBP-1), interferon alpha inducible protein (IFI)-6-16, and IFI-27, inhibit HCV subgenomic replication. In this study we investigated the effects of these ISGs against HCV in cell culture and their direct molecular interaction with viral proteins. HCV replication and virus production were suppressed significantly by overexpression of GBP-1, IFI-6-16, or IFI-27. Knockdown of the individual ISGs enhanced HCV RNA replication markedly. A two-hybrid panel of molecular interaction of the ISGs with HCV proteins showed that GBP-1 bound HCV-NS5B directly. A protein truncation assay showed that the guanine binding domain of GBP-1 and the finger domain of NS5B were involved in the interaction. Binding of NS5B with GBP-1 inhibited its guanosine triphosphatase GTPase activity, which is essential for its antiviral effect. Taken together, interferon-induced GBP-1 showed antiviral activity against HCV replication. Conclusion: Binding of the HCV-NS5B protein to GBP-1 countered the antiviral effect by inhibition of its GTPase activity. These mechanisms may contribute to resistance to innate, IFN-mediated antiviral defense and to the clinical persistence of HCV infection. (HEPATOLOGY 2009.)
Hepatitis C virus (HCV) is one of the most important pathogens causing liver-related morbidity and mortality.1 Hepatitis C is characterized by persistent infection of the liver, leading to the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Type-I interferon (IFN) plays a central role in eliminating viruses, not only by way of therapeutic applications2 but also as a natural cellular antiviral mechanism.3, 4 Interferons are produced naturally in response to virus infection and to cellular exposure to IFN itself. Binding of the IFNs to their receptors activates the Jak-STAT pathway to form a complex with IFN-stimulated gene factor-3 (ISGF3), which translocates to the nucleus, binds the IFN-stimulated response element (ISRE) located in the promoter/enhancer region of the IFN-stimulated genes (ISGs), and activates expression of ISGs.
HCV subgenomic replicons constitute in-vitro models that simulate cellular autonomous replication of HCV genomic RNA. Replication of the HCV replicon can be abolished by treatment with small amounts of type-I and type-II IFNs.5, 6 These findings suggest that various molecules encoded by the ISGs have antiviral activities against HCV replication. We have reported previously that the baseline activities of ISG expression are substantially decreased in cells expressing HCV replicon and that this decrease is partly attributable to the transcriptional suppression of interferon regulatory factor 1 (IRF-1).7 We performed expressional screening of ISGs to investigate their antiviral effects against HCV replication and showed that guanylate binding protein 1 (GBP-1), interferon alpha inducible protein (IFI)-6-16, and IFI-27 had novel activities against cells harboring an HCV replicon.8 In this study we investigated the antiviral effects and molecular mechanism of GBP-1, IFI-6-16, and IFI-27 on HCV-JFH1-infected cells.
Materials and Methods
- Top of page
- Materials and Methods
- Supporting Information
Cells and Cell Culture.
Huh7, Huh7.5.1, and 293T cells were maintained in Dulbecco's modified minimal essential medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum at 37°C under 5% CO2. To maintain cell lines carrying the HCV replicon (Huh7/Rep-Feo cells), G418 (Nacalai Tesque, Kyoto, Japan) was added to the culture medium to a final concentration of 500 μg/mL.
HCV Replicon Constructs and Transfection.
The HCV replicon plasmids, which contain Rep-Feo, were derived from the HCV-N strain, pHC1bneo/delS (1b-Feo) and HCV-JFH1 strain, pSGR-JFH1 (2a-Feo).6, 9 These constructs express a chimeric reporter protein of firefly luciferase (Fluc) and neomycin phosphotransferase. The replicon RNA synthesis and transfection have been described (Huh7/Rep-1bFeo, Huh7/Rep-2aFeo).10, 11
HCV Cell Culture System.
A plasmid, pJFH1-full,12 which encodes the full-length HCV-JFH1 sequence, was linearized and used as a template for synthesis of HCV RNA using the RiboMax Large Scale RNA Production System (Promega, Madison, WI). After DNaseI (RQ-1, RNase-free DNase, Promega) treatment, the transcribed HCV RNA was purified using ISOGEN (Nippon Gene, Tokyo, Japan). For the RNA transfection, Huh-7.5.1 cells were washed twice, and 5 × 106 cells were suspended in Opti-MEM I (Invitrogen, Carlsbad, CA) containing 10 μg of HCV RNA, transferred into a 4-mm electroporation cuvette, and subjected to an electric pulse (1,050 μF and 270 V) using the Easy Ject system (EquiBio, Middlesex, UK). After electroporation, the cell suspension was left for 5 minutes at room temperature and then incubated under normal culture conditions in a 10-mm diameter cell culture dish. The transfected cells were split every 3 to 5 days. The culture supernatants were subsequently transferred onto uninfected Huh-7.5.1 cells.
Construction of Plasmids Expressing ISGs and Analysis of Their Effect on HCV Subgenomic and Genomic Replication.
We constructed plasmids expressing GBP-1, IFI-6-16, IFI-27, and IRF-1. The full-length human ISGs were amplified by polymerase chain reaction (PCR) from Huh7 cells and cloned into pcDNA3.1D/V5-His-TOPO (pcDNA4/TO/myc-his for IRF-1) (Invitrogen) to yield the mammalian expression construct, pcDNA-ISG. The ISG-expression plasmid, pcDNA-ISG, was transfected into Huh7/Rep-1bFeo or Huh7/2aFeo cells, and the replication level of the HCV replicon was analyzed by luciferase assay. A plasmid, pcDNA3.1D/V5-His/lacZ (Invitrogen), was used as a control plasmid vector for mock transfection.
Another plasmid, pcDNA-ISG, was transfected into HCV-JFH1 cell culture systems. Forty-eight hours after transfection the culture supernatants, total cellular RNA, and protein, which were used for quantification of HCV core antigen, were harvested.
Luciferase activity was measured with a Lumat LM9501 luminometer (Promega) using a Bright-Glo Luciferase Assay System (Promega) or a Dual-Luciferase Reporter Assay System (Promega).
Real-Time Reverse Transcription (RT)-PCR Analysis.
Total cellular RNA was isolated using ISOGEN (Nippon Gene). Two micrograms of total cellular RNA were used to generate complementary DNA (cDNA) from each sample using SuperScript II (Invitrogen) reverse transcriptase. Expression of messenger RNA (mRNA) was quantified using the TaqMan Universal PCR Master Mix and the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The primers have been described.13
Quantification of HCV Core Antigen in Culture Supernatants.
Culture supernatants of JFH1-RNA transfected Huh-7.5.1 cells were collected on the days indicated, passed through a 0.45-μm filter (MILLEX-HA, Millipore, Bedford, MA), and stored at −80°C. The concentrations of core antigen in the culture supernatants were measured using a chemiluminescence enzyme immunoassay (CLEIA) according to the manufacturer's protocol (Lumipulse Ortho HCV Antigen, Ortho-Clinical Diagnostics, Tokyo, Japan).
Synthetic Short Hairpin RNA (shRNA) and shRNA-Expression Plasmid.
ISG-directed shRNA expression vectors (pUC19-shRNA-ISG) were designed and constructed as described.11 Briefly, oligodeoxyribonucleotides encoding shRNA sequences were synthesized and cloned just downstream of the human U6 promoter in the plasmid pUC19. To avoid problems of structural instability of DNA strands arising from the tight palindrome structure during transcription of shRNA, several point mutations were introduced into the sense strand of the shRNA sequences, which retained fully the silencing activity of the shRNA.11 Sequences of the shRNAs are shown in Supporting Table 1.
Construction of Plasmids Expressing Full-Length and Truncated HCV-NS Proteins.
Expression plasmids of HCV-NS3, NS4B, NS5A, and NS5B were constructed by inserting PCR-amplified fragments encoding each HCV-NS protein into pcDNA4/TO/myc-his (pcDNA-NS3, pcDNA-NS4B, pcDNA-NS5A, and pcDNA-NS5B, respectively). The plasmids, which expressed truncated HCV-NS5B proteins, were generated by insertion of various fragments amplified by PCR using pcDNA-NS5B into pcDNA4/TO/myc-his.
Plasmids expressing HCV-NS protein (pcDNA-NS3, pcDNA-NS4B, pcDNA-NS5A, or pcDNA-NS5B) and plasmids expressing ISG (pcDNA-GBP-1, pcDNA-IFI-6-16, or pcDNA-IFI-27) were cotransfected into HEK-293 T cells. Forty-eight hours after transfection, cellular proteins were harvested and immunoprecipitation assay was performed using an Immunoprecipitation Kit according to the manufacturer's protocol (Roche Applied Science, Mannheim, Germany). The immunoprecipitated proteins were analyzed by western blotting.
Mammalian Two-Hybrid Assay.
Mammalian two-hybrid assay (Stratagene, La Jolla, CA) is a method for detecting protein-protein interactions in vivo in mammalian cells. In this assay a gene encoding the HCV-NS protein (NS3, NS4B, NS5A, and NS5B) was fused to the DNA-binding domain of the yeast protein GAL4 (a bait plasmid, pcCMV-BD), whereas another gene (ISG; GBP-1, IFI-6-16, and IFI-27) was fused to the transcriptional activation domain of the mouse protein nuclear factor-kappaB (NF-κB) (an acceptor plasmid, pCMV-AD). These two-hybrid constructs are cotransfected into Huh-7 cells with a reporter plasmid encoding the firefly-luciferase gene. If the ISG protein and HCV-NS protein interact, they create a functional transcriptional activator by bringing the activation domain into close proximity with the DNA-binding domain; this can be detected by expression of the luciferase reporter gene. The ISG-encoding site was inserted into a plasmid, pCMV-AD, by cloning from a plasmid, pcDNA-ISG (pCMV-AD-ISG). The HCV-NS protein gene, which was subcloned from a plasmid, pcDNA-NS protein, was also inserted into a plasmid, pCMV-BD (pCMV-BD-NS protein). The plasmids pCMV-AD-ISG and pCMV-BD-NS protein were cotransfected with a reporter plasmid, pFR-luc encoding Fluc into Huh-7 cells. Cellular proteins were harvested after 48 hours and luciferase assays were performed.
Huh7.5.1 cells infected with HCV-JFH-1 were seeded onto 18-mm round micro-cover glasses (Matsunami, Tokyo, Japan). After transfection of plasmids expressing ISG, pcDNA-ISG, Huh7.5.1 cells were fixed with cold acetone. The cells were incubated with the primary antibodies for 1 hour at 37°C and with Alexa Flour 488 goat antimouse immunoglobulin G (IgG) antibody and Alexa 568 donkey antigoat IgG antibody (Molecular Probes, Eugene, OR) for 1 hour at room temperature. Cells were mounted with VectaShield Mounting Medium and DAPI (Vector Laboratories, Burlingame, CA) and visualized with fluorescence microscopy (BZ-8000, Keyence, Osaka, Japan).
The Establishment of a Mutant Form of GBP-1.
In order to introduce mutations into GBP-1, the full-length human ISGs were amplified by PCR from a human liver cDNA library (Invitrogen) and cloned into pCMV-Tag Epitope Tagging Mammalian Expression Vectors; pCMV-GBP-1 (Stratagene). The following mutations were introduced into the DNA fragment in the subcloning vector by site-directed mutagenesis (Quick-Change II Site-Directed Mutagenesis Kit; Stratagene): R48P; pCMV-GBP1.R48P.
Guanosine Triphosphatase (GTPase) Assay.
Cellular proteins were harvested from Huh7 cells 48 hours after transfection of a plasmid expressing HCV-NS5B, pcDNA-NS5B, or a plasmid for mock transfection, pcDNA3. A GTPase assay was performed to examine GTPase activity of cellular proteins using GTPase ELIPA kits (Cytoskelton, Denver, CO). The assay is based on an absorbance shift (340 to 360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside is catalytically converted to 2-amino-6-mercapto-7-methyl purine in the presence of inorganic phosphate (Pi). The reaction is catalyzed by purine nucleoside phosphorylase. One molecule of inorganic phosphate will yield one molecule of 2-amino-6-mercapto-7-methyl in an essentially irreversible reaction. Thus, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the reaction.
Transient transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.
Luciferase activity was measured with a Lumat LM9501 luminometer (Promega) using a Bright-Glo Luciferase Assay System (Promega).
Western Blot Analysis.
Western blotting was performed as described.10 Briefly, 10 μg of total cell lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with the primary antibodies followed by a peroxidase-labeled anti-IgG antibody and visualized by chemiluminescence using the ECL Western Blotting Analysis System (Amersham Biosciences, Buckinghamshire, UK).
Statistical analyses were performed using Student's t test; P values of less than 0.05 were considered statistically significant.
- Top of page
- Materials and Methods
- Supporting Information
GBP1, IFI-6-16, and IFI-27 Suppress HCV Subgenomic Replicon and Virus Replication in Cell Culture.
First, we assessed the effects of GBP-1, IFI-6-16, and IFI-278 on replication of HCV genotype 1b and 2a replicons.9–11 Transfection of the expression plasmids for IRF-1, GBP-1, IFI6-16, and IFI-27 resulted in expression of corresponding proteins (Fig. 1A). Each ISG-expression plasmid was transfected into cells harboring HCV genotype 1b or 2a replicons that expressed Fluc and luciferase assays were performed. Expression of GBP-1, IFI-6-16, and IFI-27 resulted in significant reduction of HCV replication (1b-Feo: GBP-1; 63.7 ± 7.49%, IFI-6-16; 62.6 ± 19.2%, IFI-27; 71.6 ± 1.22%, IRF-1; 95.1 ± 0.19% / 2a-Feo: GBP-1; 61.9 ± 12.3%, IFI-6-16; 42.9 ± 7.0%, IFI-27; 42.9 ± 13.2%, IRF-1; 95.8 ± 1.0%) (Fig. 1B).
We next evaluated whether these ISGs affected the replication of HCV-JFH1 in cell culture. Each ISG-expression plasmid was transfected into HCV-JFH1-infected Huh-7.5.1 cells. The secretion of HCV particles was measured using an HCV core antigen assay. HCV core protein in the supernatant, as well as the cellular fraction, was significantly decreased by the overexpression of GBP-1 (24.3 ± 2.90%, 35.6 ± 6.73%), IFI-6-16 (33.9 ± 0.40%, 43.6 ± 1.14%), IFI-27 (38.5 ± 3.63%, 61.2 ± 1.28%), or IRF-1 (69.2 ± 1.89%, 78.3 ± 1.01%) in a dose-dependent manner (Fig. 1C). Real-time RT-PCR showed that the expression level of HCV RNA was significantly suppressed by plasmids expressing GBP-1 (38.2 ± 1.39%), IFI-6-16 (54.6 ± 2.93%) IFI-27 (50.7 ± 2.81%), and IRF-1 (6.61 ± 0.341%) (Fig. 1D). In Fig. 1E, levels of intracellular HCV core protein were decreased but less than those of HCV-RNA, which might be attributable to the longer half-life of HCV core protein than that of HCV-RNA. To visualize the suppressive effects of ISG on HCV in cell culture, we performed immunohistochemistry on the HCV-JFH1 cell culture system after transfection of the ISG plasmids using anti-HCV core antibodies and anti-ISG protein antibodies. As shown in Fig. 2, HCV core protein expression was markedly decreased in cells in which the ISG transgene was overexpressed. As a control, a LacZ expression plasmid was transfected into the same cells and HCV core protein expression did not decrease. Taken together, GBP-1, IFI-6-16, and IFI-27 showed direct antiviral effects on HCV genomic replication and also particle formation and secretion from cultured cells.
The Effects of Knockdown of GBP-1, and IFI-6-16, and IFI-27 on the HCV-JFH Cell Culture System.
We subsequently investigated the effects of suppression of GBP-1, IFI-6-16, and IFI-27 expression on the HCV-JFH1 cell culture system. To conduct the study we used shRNA expression-plasmids, pUC19-shRNA-GBP-1, pUC19-shRNA-6-16, and pUC-shRNA-IFI-27, which expressed shRNA that targeted the corresponding genes. The shRNA-expressing plasmids were cotransfected with plasmids expressing the respective target genes into Huh7 cells. Western blotting showed that the expression levels of the target proteins were significantly suppressed by the respective shRNAs (Fig. 3A). Next, we transiently transfected shRNA-expression plasmids into HCV-JFH1-infected Huh-7.5.1 cells and performed HCV core antigen assays, real-time RT-PCR, and western blotting to measure HCV-RNA and the protein expression, respectively. Supernatant HCV core protein was significantly and dose-dependently increased by the overexpression of sh-GBP-1 (218.2 ± 88.7%), sh-IFI-6-16 (206.4 ± 74.5%), or sh-IFI27 (234.2 ± 46.3%) (Fig. 3B). In cells that were transfected with sh-GBP-1, sh-IFI-6-16, and sh-IFI27, expression of HCV and particle formation were significantly increased (161.1 ± 1.95% for shRNA-GBP-1, 155.7 ± 18.6% for shRNA-6-16, 114.6 ± 7.13% for shRNA-IFI-27; Fig. 3C). We also found by western blotting analysis that expression of HCV core protein was increased by the shRNA (Fig. 3D).
Intermolecular Interaction of the Three ISGs and HCV Nonstructural Proteins.
It has been reported that HCV structural and nonstructural (NS) proteins interact with various host proteins. ISGs such as LMP7, PKR, and 2′,5′-oligoadenylate synthase (25OAS) also are known to interact with HCV NS proteins.14, 15 We performed immunoprecipitation assays to study the direct intermolecular interaction of HCV proteins and ISGs. Plasmids expressing individual HCV-NS proteins were cotransfected with the ISG-expression plasmids into HEK-293T cells. Cellular proteins were harvested 48 hours after transfection and subjected to immunoprecipitation assay. As shown in Fig. 4, we found that GBP-1 bound to HCV-NS5B. The molecular interactions between the NS proteins and ISGs were verified by the mammalian two-hybrid assay. We transfected fusion gene plasmids that express bait and each HCV NS protein together with, as targets, the three ISGs individually cotransfected into cells with reporter plasmids. Only GBP-1 and NS5B showed a significantly increased luciferase activity that was induced by bait-target interaction (Fig. 5). These results indicated a significant and specific molecular interaction between NS5B and GBP-1.
The Analysis of the Domains of Interaction in HCV-NS5B and GBP-1.
Having shown a molecular interaction between GBP-1 and HCV-NS5B, we carried out protein truncation assays. To define interacting regions of GBP1 and NS5B, we constructed expression plasmids for the truncated proteins according to the reported functional domains, which were the GTPase and alpha-helical domains for GBP-1 and the finger, palm, and thumb domains for NS5B (Fig. 6A,B).16, 17 In Fig. 6C,D, plasmids expressing truncated proteins of NS5B or GBP-1 were transfected into HEK-293T cells. Cell lysates were used in western blotting to confirm expression of truncated proteins. These plasmids expressing truncated proteins were transfected into cells and monitored by the mammalian two-hybrid assay. After 48 hours of transfection, luciferase assays were performed to monitor molecular interaction. As shown in Fig. 6E, a significantly higher luciferase activity was obtained when the cells were transfected with full-length GBP-1 and a truncated NS5B that contained amino acids (aa) 1 though 70, which corresponded to the finger domain. Truncation of GBP-1 showed that the GTPase domain (aa 1 through 317) and full-length NS5B gave significantly higher luciferase activities (Fig. 6F). These results showed that the NS5B-finger domain and the GTPase domain of GBP-1 were the regions of mutual molecular interaction.
The GTPase Activity has a Direct Antiviral Effect on HCV.
To determine whether the GTPase activity of GBP1 was necessary for the antiviral action, we constructed a mutant GBP-1 expression plasmid in which an aa 48 P for R substitution was introduced in the catalyzing domain to abolish GTPase activity (Fig. 7A).18 As shown in Fig. 7B,C, the inactivated mutant GBP1 showed no antiviral activity. These results indicated that the GTPase activity is essential for the specific suppression of HCV replication.
HCV-NS5B Inhibits GTPase Activity and Results in Continuous Intracellular Replication of HCV.
There are several reports that HCV establishes stable infection and replication in cells by interacting with host proteins and affecting their antiviral, antiproliferative, and apoptotic activities.14, 19 Therefore, we determined whether binding of NS5B to GPB-1 affects protein expression levels or enzymatic functions. As shown in Fig. 8A, transfection of NS5B into cells significantly suppressed IFN-gamma-induced production of GBP-1 proteins. However, there were no significant differences in mRNA expression levels between cells with and without overexpression of NS5B (Fig. 8B). Expression of NS5B showed no significant effect on mRNA and protein expression levels of IFI-6-16 and IFI-27. These findings suggested that NS5B specifically suppresses GBP1 posttranslation. Next, we examined the effects of NS5B on the GTPase activity of GBP1 using a cell-free GTPase assay (Fig. 8C). Baseline cellular GTPase activity was increased by overexpression of GBP-1 or by addition of IFN-gamma. On the contrary, transfection of NS5B substantially suppressed GTPase activities of baseline and also IFN-treated cells.
Finally, we investigated whether NS5B negatively regulate antiviral activity of GBP-1 on HCV. GBP-1-expression plasmid was transfected into HCV replicon cells, Huh7/Rep-1bFeo, together with or without expression plasmid of a truncated NS5B1-70, which lacked enzymatic activity but showed molecular interaction with GBP-1 (Fig. 6E). Luciferase assays showed that the suppressive effect of GBP-1 on HCV was abolished by overexpression of NS5B1-70 (Fig. 8D). These results suggest that NS5B may substantially rescue the inhibitory effect of GBP-1 on HCV replication.
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- Materials and Methods
- Supporting Information
Persistence of virus replication in host cells is governed by the cellular antiviral system and by the ability of the virus to evade antiviral responses.20 In this study we demonstrated previously undescribed antiviral activities of the three ISGs, GBP-1, IFI-6-16, and IFI-27, against HCV-JFH1 replication and release of virus particles (Figs. 1, 2). GBP-1 is a well-described GTPase.21 A mutation in the GTPase-catalyzing domain completely abolished antiviral activity against HCV (Fig. 7B,C), suggesting that GTPase activity is essential for antiviral action. Furthermore, we have shown specific binding of GBP-1 and HCV-NS5B, involving the GBP-1 guanine-binding domain and the NS5B finger domain (Fig. 6). Interestingly, overexpression of NS5B substantially suppressed the GTPase activity of host cellular GTPases, including GBP-1, and rescued the antiviral action of GBP-1 onto HCV (Fig. 8). Taken together, the interplay between the viral NS5B and host GBP-1 protein might be involved in the persistence of HCV infection and its resistance to IFN.
GBP-1 belongs to a group of GTPase families, including the p47 GBP family, the p65 GBP family, Mx, and very large inducible GTPase.22–25 The p65 GBP family has five members, GBP-1 to GBP-5.26–29 There is one report that GBP-1 mediates antiviral effects against vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) in HeLa cells.30 In our previous study, overexpression of GBP-1 significantly suppressed replication of an HCV subgenomic replicon and suppression of GBP-1 caused the HCV replication level to increase. In HCV-JFH1-infected cells we found that overexpression of GBP-1 significantly reduced the formation of HCV particles and the intracellular replication level of HCV RNA, and that shRNA-directed suppression of GBP-1 resulted in an increase of HCV particles and HCV RNA. These findings suggest that GBP-1 may suppress intracellular HCV replication directly and that the expression of GBP-1 in the absence of IFN treatment may result in inhibition of virus replication. However, little is known about the underlying antiviral mechanisms. Our previous study showed that GBP-1 had no effect on the translational activity of the HCV internal ribosome entry site (IRES) and intracellular signaling pathways such as ISRE, AP-1, GAS, and NF-κB.8
GBP-1 has functional homology with MxA, which shows GTPase activity and mediates degradation of cellular RNA, general repression of protein synthesis, and apoptotic cell death.21 The GTPase domain of GBP-1 is able to bind GMP, GDP, and GTP with similar affinities and to hydrolyze GTP to GMP.16, 31–33 Analyses of the crystal structure of GBP-1 have shown that it has a 36-kDa N-terminal large G (LG) domain (residues 1-317), which retains the main biochemical properties of GBP-1, and an elongated 32 kDa carboxy-terminal alpha-helical domain.16 In our study we demonstrated that the antiviral activity of GBP-1 required the GTPase activity that is the main biochemical property of GBP-1. We highlight the GBP-1 mutant, GBP-1.R48P, which exhibits no detectable GTP binding or hydrolysis and acts as a dominant negative mutant.34 Overexpression of GBP-1.R48P had no antiviral effect on HCV replication in cells expressing the HCV subgenomic replicon or replicating HCV-JFH1. These results suggest that the GTPase activity of GBP-1 has an antiviral action against HCV replication. It has been reported that IFNs induced translocation of GBP-1 proteins from the cytoplasm to the Golgi complex.18 We expect that GBP-1 proteins play some role in inhibiting the HCV life cycle.
It has been reported that some HCV proteins interact with various host proteins. Among the ISGs, LMP7, PKR, and 25OAS interacted with HCV-NS proteins.14, 15, 19 We focused on the interaction of ISG proteins with HCV-NS proteins using immunoprecipitation and mammalian two-hybrid assays. We found that GBP-1 bound to NS5B. Furthermore, we revealed that the molecular regions responsible were the LG domain of GBP-1 and the finger domain of NS5B. It was suggested that GBP-1 suppresses the biochemical activity of NS5B, the RNA-dependent RNA polymerase, or that NS5B affected the GTPase of GBP-1 to enable persistent infection by HCV. We performed cotransfection of GBP-1-expression plasmids with plasmids expressing NS5B into Huh7 cells to analyze these interactions. In western blotting analysis we found that the level of GBP-1 protein was reduced by HCV-NS5B. We confirmed that the biochemical properties of GBP-1 proteins were suppressed by NS5B proteins in GTPase assays. These results suggest that HCV-NS5B has a suppressive effect on the GTPase activity of GBP-1, resulting in persistent infection and replication of HCV.
Several reports suggest that viruses target the IFN system of the host cells to establish replication.35 The IRF-3-mediated IFN-beta induction pathway could be a target for viruses to counteract antiviral responses and promote their replication. Ebola virus, bovine viral diarrhea virus (BVDV), and influenza A virus interfere with the activation of IRF-3 through the activities of their virus-encoded proteins.36–38 It was recently reported that the HCV NS34A protease blocks virus-induced activation of IRF-3, possibly by proteolytic cleavage of Cardif.39, 40 We recently reported that the double-stranded RNA-triggered and RIG-I-triggered IFN expression and this blockade was partly mediated by NS4B.41 These reports suggest that HCV-NS proteins are involved in the persistence of intracellular replication and infection of HCV. In the present study we found that NS5B bound to GBP-1 and blocked its GTPase activity, the main biological property of GBP-1 to cause HCV replication and infection. Accordingly, we suggest that NS5B has RNA-dependent RNA polymerase activity but also an effect on antiviral host defense.
In conclusion, we found novel antiviral effects of GBP-1, IFI-6-16, and IFI-27 on the HCV life cycle. GBP-1 interacted with HCV-NS5B, but NS5B has suppressive activities on GBP-1 to establish persistent infection and intracellular replication.
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- Materials and Methods
- Supporting Information
We thank Mr. Satoshi Yamazaki at JST, ERATO, Nakauchi Stem Cell and Organ Regeneration Project for excellent technical assistance.
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- Materials and Methods
- Supporting Information
- 1Epidemiology of hepatitis C. HEPATOLOGY 1997; 26: 62S–65S..
- 2Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 1998; 339: 1485–1492., , , , , , et al.
- 3Antiviral actions of interferons. Clin Microbiol Rev 2001; 14: 778–809..
- 4The interferon-alpha/beta system in antiviral responses: a multimodal machinery of gene regulation by the IRF family of transcription factors. Curr Opin Immunol 2002; 14: 111–116., .
- 5Interferon-gamma inhibits replication of subgenomic and genomic hepatitis C virus RNAs. HEPATOLOGY 2002; 35: 694–703., , , , , , et al.
- 6Effect of alpha interferon on the hepatitis C virus replicon. J Virol 2001; 75: 8516–8523., , .
- 7Regulation of hepatitis C virus replication by interferon regulatory factor-1. J Virol 2004; 78: 9713–9720., , , , , , et al.
- 8Expressional screening of interferon-stimulated genes for antiviral activity against hepatitis C virus replication. J Viral Hepat 2006; 13: 690–700., , , , , , et al.
- 9Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 2003; 125: 1808–1817., , , , , , et al.
- 10Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon-alpha. J Infect Dis 2004; 189: 1129–1139., , , , , , et al.
- 11Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs. EMBO Rep 2003; 4: 602–608., , , , , , et al.
- 12Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005; 11: 791–796., , , , , , et al.
- 13Development of plaque assays for hepatitis C virus and isolation of mutants with enhanced cytopathogenicity and replication capacity. Virology 2008; 371: 71–85., , , , , , et al.
- 14Hepatitis C virus non-structural protein NS3 interacts with LMP7, a component of the immunoproteasome, and affects its proteasome activity. Biochem J 2004; 384: 401–409., , , , .
- 15Hepatitis C virus NS5A protein interacts with 2',5'-oligoadenylate synthetase and inhibits antiviral activity of IFN in an IFN sensitivity-determining region-independent manner. J Gen Virol 2004; 85: 959–969., , , , , , et al.
- 16How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP. Nature 2006; 440: 101–104., , , , .
- 17Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat Struct Biol 1999; 6: 937–943., , , , , .
- 18Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor. Proc Natl Acad Sci U S A 2005; 102: 8680–8685., , .
- 19Antiapoptotic and oncogenic potentials of hepatitis C virus are linked to interferon resistance by viral repression of the PKR protein kinase. J Virol 1999; 73: 6506–6516., , , , .
- 20To interfere and to anti-intefere: the interplay between hepatitis C virus and interferon. Viral Immunol 2002; 15: 95–119., .
- 21How cells respond to interferons. Annu Rev Biochem 1998; 67: 227–264., , , , .
- 22Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins. Nature 2000; 403: 567–571., , , , .
- 23IFN-inducible GTPases and immunity to intracellular pathogens. Trends Immunol 2004; 25: 601–609..
- 24Interferon-induced mx proteins: dynamin-like GTPases with antiviral activity. Traffic 2002; 3: 710–717., .
- 25A giant GTPase, very large inducible GTPase-1, is inducible by IFNs. J Immunol 2003; 171: 1255–1265., , , , .
- 26Two families of GTPases dominate the complex cellular response to IFN-gamma. J Immunol 1998; 161: 6715–6723., , , , , , et al.
- 27Murine GBP-2: a new IFN-gamma-induced member of the GBP family of GTPases isolated from macrophages. J Interferon Cytokine Res 1998; 18: 977–985., , , , , , et al.
- 28Cloning, expression, and characterization of a novel guanylate-binding protein, GBP3 in murine erythroid progenitor cells. Biochim Biophys Acta 1998; 1384: 373–386., , , , .
- 29Murine GBP-5, a new member of the murine guanylate-binding protein family, is coordinately regulated with other GBPs in vivo and in vitro. J Interferon Cytokine Res 2002; 22: 899–909., , , , .
- 30Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus. Virology 1999; 256: 8–14., , , , .
- 31The interferon-induced 67-kDa guanylate-binding protein (hGBP1) is a GTPase that converts GTP to GMP. J Biol Chem 1994; 269: 11299–11305., .
- 32Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif. J Mol Biol 1999; 292: 321–332., , , , .
- 33GTPase properties of the interferon-induced human guanylate-binding protein 2. FEBS Lett 1996; 390: 69–72., , , .
- 34Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis. J Mol Biol 2004; 344: 257–269., , , , , .
- 35Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 2006; 441: 101–105., , , , , , et al.
- 36The Ebola virus VP35 protein inhibits activation of interferon regulatory factor 3. J Virol 2003; 77: 7945–7956., , , , , , et al.
- 37Noncytopathic bovine viral diarrhea virus inhibits double-stranded RNA-induced apoptosis and interferon synthesis. J Virol 2001; 75: 4692–4698., .
- 38Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein. J Virol 2000; 74: 7989–7996., , , , , , et al.
- 39Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 2003; 300: 1145–1148., , , , , , et al.
- 40Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 2005; 437: 1167–1172., , , , , , et al.
- 41Hepatitis C virus non-structural proteins responsible for suppression of the RIG-I/Cardif-induced interferon response. J Gen Virol 2007; 88: 3323–3333., , , , , , et al.
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Additional Supporting Information may be found in the online version of this article.
|HEP_23195_sm_SuppTab.doc||20K||Supporting Table 1. Sequences of the shRNAs for mRNA knock down analyses.|
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