Guanylate-binding protein 1 participates in cellular antiviral response to dengue virus
© Pan et al.; licensee BioMed Central Ltd. 2012
Received: 14 May 2012
Accepted: 15 November 2012
Published: 27 November 2012
Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus found in tropical and sub-tropical regions around the world. Vaccines against DENV are currently unavailable. Guanylate-binding protein 1 (GBP1) is one of the Interferon (IFN) stimulated genes (ISGs) and has been shown important for host immune defense against various pathogens. However, the role of GBP1 during DENV infection remains unclarified. In this study, we evaluated the relevance of GBP1 to DENV infection in in vitro model.
Quantitative RT-PCR (qRT-PCR) and Western blot showed that the expression of mouse Gbp1 was dramatically upregulated in DENV-infected RAW264.7 cells. The intracellular DENV loads were significantly higher in Gbp1 silenced cells compared with controls. The expression levels of selective anti-viral cytokines were decreased in Gbp1 siRNA treated cells, while the transcription factor activity of NF-κB was impaired upon GBP1 silencing during infection.
Our data suggested that GBP1 plays an antiviral role during DENV infection.
KeywordsGBP1 DENV Antiviral response NF-κB
Dengue virus (DENV), a member of the mosquito-borne flavivirus family, is an icosahedral, enveloped virus with a single-stranded positive sense RNA genome. Millions of cases of DENV infection occur worldwide each year [1, 2]. Dengue hemorrhagic fever, the severe form of DENV infections, can cause serious haemorrhage, sudden drop in blood pressure (shock) and even death. Since no vaccine against DENV is currently available, much effort is needed to explore the host antiviral mechanisms for control of DENV infection and vaccine development [1, 3–5].
Interferons (IFNs) are major antiviral cytokines released by host cells in response to viral and other pathogenic infections and play crucial roles in induction and regulation of both innate and adaptive immune responses . IFNs establish an antiviral state by activating mainly the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway and other signaling cascades. Even though hundreds of genes are characterized as interferon-stimulated genes (ISGs), the roles of many ISGs during infection remain largely unknown [7, 8].
Guanylate-binding protein 1 (GBP1) is one of the ISGs that most strongly induced by IFNs  and belongs to a family of GTPases which are divided into three groups: (1) the large GTPases, also known as GBPs; (2) the small GTPases; (3) the Mx proteins. The human large GTPase family is composed of seven members encoded by a gene cluster located on chromosome 1 [10, 11]. The emerging roles of GBP1 in host immune responses have been characterized in in vitro and in vivo models [12–15]. For example, GBP1 is overexpressed in endothelial cells upon activation of inflammatory cytokines and is involved in intestinal mucosal inflammation . Recently, a study using Gbp1 knockout mice indicated that GBP1 has strong anti-bacterial activity . There are also reports suggesting that GBP1 is involved in host immune response against chlamydia  and viruses including vesicular stomatitis virus, encephalomyocarditis virus and Hepatitis C Virus [19–21]. However, the distinct role of GBP1 during infection of other pathogens, including mosquito-borne flaviviruses remains largely unknown. We hereby investigated the physiological role of GBP1 during DENV infection in different in vitro models.
Gbp1 is upregulated upon DENV infection
Gbp1 has antiviral activity against DENV infection
siRNA and oligo-primer sequences for this study
siRNA seq for mouse gene Gbp1
siRNA seq for human gene GBP1
Forward primer for qRT-PCR of mouse beta-actin
Reverse primer for qRT-PCR of mouse beta-actin
Forward primer for qRT-PCR of human beta-actin
Reverse primer for qRT-PCR of human beta-actin
Forward primer for qRT-PCR of DENV E gene
Reverse primer for qRT-PCR of DENV E gene
Forward primer for qRT-PCR of mouse Gbp1 gene
Reverse primer for qRT-PCR of mouse Gbp1 gene
Forward primer for qRT-PCR of human GBP1 gene
Reverse primer for qRT-PCR of human GBP1 gene
Selective cytokines are downregulated in Gbp1 silenced cells upon DENV infection
Transcriptional factor activity of NF-ÎºB is impaired in GBP1 silenced cells
More and more attention has been focused on the roles of large GTPase family in immune response [12–14]. GBP1 can be induced by IFNγ as well as IFNα/β, and its induction can be augmented by TNF-α, IL-1 or LPS. The inhibitory roles of GBP1 to different pathogens have been reported [17–19, 21]. Itsui and his colleague showed that HCV replication was suppressed significantly by overexpression of GBP1, while binding of the HCV-NS5B protein to GBP1 countered the antiviral effect through inhibition of GTPase activity . MacMicking speculated that GBP1 might help to limit the cell-to-cell spread of progeny virus through its anti-cell proliferative activity [13, 28]. GBP1 has modest antiviral activity against the negative strand RNA viruses (such as Rhabdovirus, vesicular stomatitis virus), as well as the positive strand RNA viruses (Picornovirus and encephalomyocarditis virus) in cultured cells . In both anti- chlamydia and antiviral study, the GTPase domain of GBP1 was suggested to be critical for its inhibitory activity [18, 21]. A recent in vivo study further suggested that GBPs can solicit host defense proteins, including the phagocyte oxidase, antimicrobial peptides, and autophagy effectors, to kill intracellular bacteria . While, more work are still needed to address the role and mechanisms of GBP1 in different infection models especially for viruses.
In this study, we confirmed that GBP1 shows inhibitory effect on DENV infection, influences the activity of NF-κB and further contributes to the production of anti-viral and pro-inflammatory cytokine/chemokines. This could be a novel mechanism for GBP1 to exert its antiviral activity in cellular level. Since NF-κB can be activated upon a broad range of pathogen infection, this could be a common action for GBP1 to play its role in different infection models. Further experiments will address the detail mechanisms of GBP1 on influencing NF-κB pathway in various models.
JD, WP and XS designed the experiments and prepared the manuscript. XZ, WP and TF performed all the experiments. All authors read and approved the final manuscript.
Type I interferon
Chemokine (C-X-C motif) ligand 1
Chemokine (C-X-C motif) ligand 2
Chemokine (C-C motif) ligand 5
C-C chemokine receptor type 5
Nuclear factor kappa B
Activator protein 1.
This work was supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (IRT1075), National Natural Science Foundation of China (NSFC) (81172812, 81271792, 31200648) and Jiangsu Natural Science Foundation (BK2012180). We thank Prof. Zhenyou Jiang from Jinan University, Guangzhou, China for technical support.
- Whitehorn J, Simmons CP: The pathogenesis of dengue. Vaccine 2011, 29: 7221-7228. 10.1016/j.vaccine.2011.07.022PubMedView ArticleGoogle Scholar
- Gubler DJ: Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol 2002, 10: 100-103. 10.1016/S0966-842X(01)02288-0PubMedView ArticleGoogle Scholar
- Murphy BR, Whitehead SS: Immune response to dengue virus and prospects for a vaccine. Annu Rev Immunol 2011, 29: 587-619. 10.1146/annurev-immunol-031210-101315PubMedView ArticleGoogle Scholar
- Fernandez-Garcia MD, Mazzon M, Jacobs M, Amara A: Pathogenesis of flavivirus infections: using and abusing the host cell. Cell Host Microbe 2009, 5: 318-328. 10.1016/j.chom.2009.04.001PubMedView ArticleGoogle Scholar
- Durbin AP, Schmidt A, Elwood D, Wanionek KA, Lovchik J, Thumar B, Murphy BR, Whitehead SS: Heterotypic dengue infection with live attenuated monotypic dengue virus vaccines: implications for vaccination of populations in areas where dengue is endemic. J Infect Dis 2011, 203: 327-334. 10.1093/infdis/jiq059PubMedPubMed CentralView ArticleGoogle Scholar
- Malmgaard L: Induction and regulation of IFNs during viral infections. J Interferon Cytokine Res 2004, 24: 439-454. 10.1089/1079990041689665PubMedView ArticleGoogle Scholar
- Rawlings JS, Rosler KM, Harrison DA: The JAK/STAT signaling pathway. J Cell Sci 2004, 117: 1281-1283. 10.1242/jcs.00963PubMedView ArticleGoogle Scholar
- Platanias LC: Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 2005, 5: 375-386. 10.1038/nri1604PubMedView ArticleGoogle Scholar
- Cheng YS, Colonno RJ, Yin FH: Interferon induction of fibroblast proteins with guanylate binding activity. J Biol Chem 1983, 258: 7746-7750.PubMedGoogle Scholar
- Prakash B, Praefcke GJ, Renault L, Wittinghofer A, Herrmann C: Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins. Nature 2000, 403: 567-571. 10.1038/35000617PubMedView ArticleGoogle Scholar
- Olszewski MA, Gray J, Vestal DJ: In silico genomic analysis of the human and murine guanylate-binding protein (GBP) gene clusters. J Interferon Cytokine Res 2006, 26: 328-352. 10.1089/jir.2006.26.328PubMedView ArticleGoogle Scholar
- Shenoy AR, Kim BH, Choi HP, Matsuzawa T, Tiwari S, MacMicking JD: Emerging themes in IFN-gamma-induced macrophage immunity by the p47 and p65 GTPase families. Immunobiology 2007, 212: 771-784.PubMedPubMed CentralView ArticleGoogle Scholar
- MacMicking JD: IFN-inducible GTPases and immunity to intracellular pathogens. Trends Immunol 2004, 25: 601-609. 10.1016/j.it.2004.08.010PubMedView ArticleGoogle Scholar
- Naschberger E, Bauer M, Sturzl M: Human guanylate binding protein-1 (hGBP-1) characterizes and establishes a non-angiogenic endothelial cell activation phenotype in inflammatory diseases. Adv Enzyme Regul 2005, 45: 215-227. 10.1016/j.advenzreg.2005.02.011PubMedView ArticleGoogle Scholar
- Lubeseder-Martellato C, Guenzi E, Jorg A, Topolt K, Naschberger E, Kremmer E, Zietz C, Tschachler E, Hutzler P, Schwemmle M, et al.: Guanylate-binding protein-1 expression is selectively induced by inflammatory cytokines and is an activation marker of endothelial cells during inflammatory diseases. Am J Pathol 2002, 161: 1749-1759. 10.1016/S0002-9440(10)64452-5PubMedPubMed CentralView ArticleGoogle Scholar
- Schnoor M, Betanzos A, Weber DA, Parkos CA: Guanylate-binding protein-1 is expressed at tight junctions of intestinal epithelial cells in response to interferon-gamma and regulates barrier function through effects on apoptosis. Mucosal Immunol 2009, 2: 33-42. 10.1038/mi.2008.62PubMedPubMed CentralView ArticleGoogle Scholar
- Kim BH, Shenoy AR, Kumar P, Das R, Tiwari S, MacMicking JD: A family of IFN-gamma-inducible 65-kD GTPases protects against bacterial infection. Science 2011, 332: 717-721. 10.1126/science.1201711PubMedView ArticleGoogle Scholar
- Tietzel I, El-Haibi C, Carabeo RA: Human guanylate binding proteins potentiate the anti-chlamydia effects of interferon-gamma. PLoS One 2009, 4: e6499. 10.1371/journal.pone.0006499PubMedPubMed CentralView ArticleGoogle Scholar
- Anderson SL, Carton JM, Lou J, Xing L, Rubin BY: Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus. Virology 1999, 256: 8-14. 10.1006/viro.1999.9614PubMedView ArticleGoogle Scholar
- Itsui Y, Sakamoto N, Kurosaki M, Kanazawa N, Tanabe Y, Koyama T, Takeda Y, Nakagawa M, Kakinuma S, Sekine Y, et al.: Expressional screening of interferon-stimulated genes for antiviral activity against hepatitis C virus replication. J Viral Hepat 2006, 13: 690-700. 10.1111/j.1365-2893.2006.00732.xPubMedView ArticleGoogle Scholar
- Itsui Y, Sakamoto N, Kakinuma S, Nakagawa M, Sekine-Osajima Y, Tasaka-Fujita M, Nishimura-Sakurai Y, Suda G, Karakama Y, Mishima K, et al.: Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein. Hepatology 2009, 50: 1727-1737. 10.1002/hep.23195PubMedView ArticleGoogle Scholar
- Dai J, Pan W, Wang P: ISG15 facilitates cellular antiviral response to dengue and west nile virus infection in vitro. Virol J 2011, 8: 468. 10.1186/1743-422X-8-468PubMedPubMed CentralView ArticleGoogle Scholar
- Tolfvenstam T, Lindblom A, Schreiber MJ, Ling L, Chow A, Ooi EE, Hibberd ML: Characterization of early host responses in adults with dengue disease. BMC Infect Dis 2011, 11: 209. 10.1186/1471-2334-11-209PubMedPubMed CentralView ArticleGoogle Scholar
- Hoang LT, Lynn DJ, Henn M, Birren BW, Lennon NJ, Le PT, Duong KT, Nguyen TT, Mai LN, Farrar JJ, et al.: The early whole-blood transcriptional signature of dengue virus and features associated with progression to dengue shock syndrome in Vietnamese children and young adults. J Virol 2010, 84: 12982-12994. 10.1128/JVI.01224-10PubMedPubMed CentralView ArticleGoogle Scholar
- Pascual G, Glass CK: Nuclear receptors versus inflammation: mechanisms of transrepression. Trends Endocrinol Metab 2006, 17: 321-327. 10.1016/j.tem.2006.08.005PubMedView ArticleGoogle Scholar
- Ivashkiv LB: Inflammatory signaling in macrophages: transitions from acute to tolerant and alternative activation states. Eur J Immunol 2011, 41: 2477-2481. 10.1002/eji.201141783PubMedPubMed CentralView ArticleGoogle Scholar
- Diamond MS, Edgil D, Roberts TG, Lu B, Harris E: Infection of human cells by dengue virus is modulated by different cell types and viral strains. J Virol 2000, 74: 7814-7823. 10.1128/JVI.74.17.7814-7823.2000PubMedPubMed CentralView ArticleGoogle Scholar
- Guenzi E, Topolt K, Cornali E, Lubeseder-Martellato C, Jorg A, Matzen K, Zietz C, Kremmer E, Nappi F, Schwemmle M, et al.: The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines. EMBO J 2001, 20: 5568-5577. 10.1093/emboj/20.20.5568PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.