Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India
Correspondence
P. Rangarajan
pnrangarajan{at}yahoo.com
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ABSTRACT |
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INTRODUCTION |
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The family Flaviviridae comprises Japanese encephalitis (JE) virus, West Nile virus, yellow fever virus, dengue virus types 1 to 4, tick-borne encephalitis virus and St Louis encephalitis virus (Kuno et al., 1998). Cellular genes such as superoxide dismutase (Liao et al., 2002
), mitogen-activated protein kinase and nuclear factor-
B (Su et al., 2002
) are activated in JEV-infected cells in culture. However, very little information is available on host gene expression changes induced by flaviviruses in the CNS. In this study, using subtraction hybridization, we have identified nine genes whose expression is upregulated by JEV in mouse brain. We further demonstrate that all these genes are also induced in the CNS by RV. Four of the genes identified in this study are known to be upregulated in the CNS by SINV, an alphavirus. Thus, common host cell pathways are activated during infection of CNS by different neurotropic viruses.
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METHODS |
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RNA isolation and analysis.
Total RNA was isolated from frozen brain tissues using TRIzol reagent (Invitrogen), and poly(A)+ RNA was isolated using an Oligotex mRNA kit (Qiagen). Northern blot analysis was performed on total brain RNA isolated from normal, saline- or virus-injected mice. RNA (20 µg) was size-fractionated on 1·2 % formaldehyde/agarose gels and transferred to nylon membranes. Radiolabelled cDNA probes were prepared using the Random-primed DNA labelling kit (Amersham Pharmacia). After hybridization and washing, Northern blots were subjected to autoradiography. All blots were first hybridized with a probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to monitor RNA loading and then rehybridized with a probe for the gene encoding the JEV envelope protein (JEVEnv) or RV surface glycoprotein to monitor virus load. JEVEnv gene expression was also analysed by RT-PCR using JEVEnv-specific primers (Ashok & Rangarajan, 1999).
Subtraction hybridization.
This was performed using the PCR-Select cDNA Subtraction kit (Clontech) essentially as per the manufacturer's instructions. Poly(A)+ RNA (2 µg) isolated from mice injected i.c. with saline or JEV and sacrificed 6 days later was used as driver and tester respectively. The differentially expressed cDNAs were cloned into pT-Adv vector (Clontech) using the AdvanTAge PCR cloning kit (Clontech). Plasmid DNA was isolated from 100 transformants using the Montage Plasmid Miniprep96 kit (Millipore) and the recombinant clones carrying inserts were identified by EcoRI digestion of the plasmids. The identity of the genes was established by DNA sequencing and searching the GenBank nucleotide database.
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RESULTS |
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DISCUSSION |
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Amongst the CNS genes upregulated by JEV, STAT1 is a transcription factor involved in the interferon response pathway and is known to be upregulated during infection by several viruses (Darnell, 1997). Cathepsin S, a lysosomal cysteine protease, is involved in the processing of MHC class II-associated invariant chain in interferon-
-stimulated microglia (Gresser et al., 2001
). Recently, cathepsins were shown to be involved in virus disassembly as well (Ebert et al., 2002
). GARG49 is a member of the recently identified glucocorticoid-attenuated response gene (GARG) family, which also includes GARG16 and GARG39 (Smith & Herschman, 1996
). GARGs are lipopolysaccharide- and interferon-inducible immediate-early/primary response genes containing multiple tetratricopeptide repeat domains, which are involved in proteinprotein interactions in a variety of cellular contexts such as mitosis-dependent ubiquitination of cyclin B, transcriptional repression, protein import into mitochondria, etc. (Goebl & Yanagida, 1991
; Lamb et al., 1995
; Tzamarias & Struhl, 1995
). The physiological significance of activation of GARGs in the CNS during virus infection is not clear. The interferon-inducible OAS produces 2',5'-oligoadenylate, which activates an endogenous ribonuclease involved in the degradation of cellular and viral RNA and leads to reduced protein synthesis (Bonnevie-Nielsen, 1990
). While three different isoforms of OAS (OAS1, OAS2 and OAS3) have been identified in humans, the cDNAs so far isolated and characterized in other species, including mouse, rat, pig and chicken, appear to correspond most closely to the OAS1 isoform (Rebouillat & Hovanessian, 1999
; Samuel, 2001
). In a recent study (Perelygin et al., 2002
), mouse strains susceptible to flavivirus infection were shown to produce a truncated form of OAS that is catalytically inactive. Further, a nonsense mutation in the gene encoding the OAS/L1 isoform was shown to be associated with West Nile virus susceptibility in laboratory mice (Mashimo et al., 2002
; Samuel, 2002
). Thus, the OAS family of genes appears to play an important role in host susceptibility to virus infection and disease. Ly-6A/E is a lymphocyte activation molecule whose expression is inducible by interferon-
in cultured glial and neuronal cells (Cray et al., 1990
). Products of the Ly-6A/E locus encode several low molecular mass cell surface proteins involved in cell signalling and/or cell adhesion processes as well as CD4+ T helper cell proliferation in response to an antigen (Henderson et al., 2002
). Thus, upregulation of Ly-6A in the brain during virus infection could be part of the host immune response mounted against the virus. Mpa2 was originally identified as an interferon-
-inducible gene expressed during the process of macrophage activation (Wynn et al., 1991
) and its exact function is not known. Thus, STAT1, OAS, cathepsin S, GARG49, Ly-6A and Mpa2 are interferon inducible-genes upregulated in the CNS during virus infection and may be involved in the host antivirus response.
ApoD is known to be induced in the brain not only during virus infections (Prosniak et al., 2001; Johnston et al., 2001
) but also during several neuropathological conditions such as schizophrenia (Thomas et al., 2001a
), Alzheimer's disease, (Thomas et al., 2001b
) etc. It is considered to be an acute phase protein involved in the removal of lipids during nerve cell degeneration and provision of lipids during the regenerative phase (Reindl et al., 2001
). Members of the Egr family of transcription factors are induced by neurotransmitters and neurotrophins and they stimulate the production of many growth factors and cytokines at the site of local tissue injury (Levkovitz & Baraban, 2002
). Egr3, another member of the Egr family of proteins was also shown to be induced in the CNS by RV (Prosniak et al., 2001
). Thus, the high virus load on day 6 p.i. may result in tissue injury leading to activation of Egr expression. P5N is involved in the hydrolysis of dCMP and UMP and its expression is induced several-fold during erythropoiesis (Hokari et al., 1998
). Thus, upregulation of P5N may result in an increase in the cellular mononucleotide pool, which may facilitate virus replication in the brain.
Of the JEV-induced CNS genes identified in this study, Mpa2, Egr1, OAS, Ly-6A and P5N have not been reported to be induced by any other neurotropic virus. Therefore we examined their expression in the CNS during RV infection. The results indicate that all the CNS genes induced by JEV are induced by RV as well. Thus, we have identified seven new RV-inducible CNS genes in this study which have not been reported earlier (Prosniak et al., 2001). As observed in the case of JEV infection, expression of cathepsin S, GARG49, P5N and OAS is induced early during RV infection when the virus load in the brain is very low, further confirming that these are likely to be early response genes. Finally, we conclude that many CNS genes are activated by more than one virus (Table 2
) suggesting that common host cell pathways are activated in the CNS during infection by different neurotropic viruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ashok, M. S. & Rangarajan, P. N. (2000). Evaluation of the potency of Biken inactivated Japanese encephalitis vaccine and DNA vaccines in an intracerebral Japanese encephalitis virus challenge. Vaccine 19, 155157.[CrossRef][Medline]
Ashok, M. S. & Rangarajan, P. N. (2002). Protective efficacy of a plasmid DNA encoding Japanese encephalitis virus envelope protein fused to tissue plasminogen activator signal sequences: studies in a murine intracerebral virus challenge model. Vaccine 20, 15631570.[CrossRef][Medline]
Biswas, S., Reddy, G. S., Srinivasan, V. A. & Rangarajan, P. N. (2001). Pre-exposure efficacy of a novel combination DNA and inactivated rabies virus vaccine. Hum Gene Ther 12, 19171922.[CrossRef][Medline]
Bonnevie-Nielsen, V. (1990). Interferon, oligoadenylate synthetase and oligoadenine nucleotide a cell biological triad. Ugeskr Laeger 152, 11401143.[Medline]
Chang, Y. E. & Laimins, L. A. (2000). Microarray analysis identifies interferon-inducible genes and Stat-1 as major transcriptional targets of human papillomavirus type 31. J Virol 74, 41744182.
Cray, C., Keane, R. W., Malek, T. R. & Levy, R. B. (1990). Regulation and selective expression of Ly-6A/E, a lymphocyte activation molecule, in the central nervous system. Brain Res Mol Brain Res 8, 915.[Medline]
Darnell, J. E., Jr (1997). STATs and gene regulation. Science 277, 16301635.
Ebert, D. H., Deussing, J., Peters, C. & Dermody, T. S. (2002). Cathepsin L and cathepsin B mediate reovirus disassembly in murine fibroblast cells. J Biol Chem 277, 2460924617.
Geiss, G. K., An, M. C., Bumgarner, R. E., Hammersmark, E., Cunningham, D. & Katze, M. G. (2001a). Global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events. J Virol 75, 43214331.
Geiss, G. K., Bumgarner, R. E., An, M. C. & 7 other authors (2001b). Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays. Virology 266, 816.
Goebl, M. & Yanagida, M. (1991). The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci 16, 173177.[CrossRef][Medline]
Gresser, O., Weber, E., Hellwig, A., Riese, S. & Regnier-Vigouroux, A. (2001). Immunocompetent astrocytes and microglia display major differences in the processing of the invariant chain and in the expression of active cathepsin L and cathepsin S. Eur J Immunol 31, 18131824.[CrossRef][Medline]
Henderson, S. C., Kamdar, M. M. & Bamezai, A. (2002). Ly-6A.2 expression regulates antigen-specific CD4+ T cell proliferation and cytokine production. J Immunol 168, 118126.
Hokari, S., Miyazaki, T., Hasegawa, M. & Komoda, T. (1998). Enhanced activity of pyrimidine 5'-nucleotidase in rat red blood cells during erythropoiesis. Biol Chem 379, 329333.[Medline]
Johnston, C., Jiang, W., Chu, T. & Levine, B. (2001). Identification of genes involved in the host response to neurovirulent alphavirus infection. J Virol 75, 1043110445.
Kuno, G., Chang, G. J., Tsuchiya, K. R., Karabatsos, N. & Cropp, C. B. (1998). Phylogeny of the genus Flavivirus. J Virol 72, 7383.
Lamb, J. R., Tugendreich, S. & Hieter, P. (1995). Tetratricopeptide repeat interactions: to TPR or not to TPR? Trends Biochem Sci 20, 257259.[CrossRef][Medline]
Levkovitz, Y. & Baraban, J. M. (2002). A dominant negative Egr inhibitor blocks nerve growth factor-induced neurite outgrowth by suppressing c-Jun activation: role of an Egr/c-Jun complex. J Neurosci 15, 38453854.
Liao, S. L., Raung, S. L. & Chen, C. J. (2002). Japanese encephalitis virus stimulates superoxide dismutase activity in rat glial cultures. Neurosci Lett 324, 133136.[CrossRef][Medline]
Manger, I. D. & Relman, D. M. (2000). How the host sees' pathogens: global gene expression responses to infection. Curr Opin Immunol 12, 215218.[CrossRef][Medline]
Mashimo, T., Lucas, M., Simon-Chazottes, D., Frenkiel, M.-P., Montagutelli, X., Ceccaldi, P. E., Deubel, V., Guenet, J.-L. & Despres, P. (2002). A nonsense mutation in the gene encoding 2'-5'-oligoadenylate synthetase/L1 isoform is associated with West Nile virus susceptibility in laboratory mice. Proc Natl Acad Sci U S A 99, 1131111316.
Mossman, K. L., Macgregor, P. F., Rozmus, J. J., Goryachev, A. B., Edwards, A. M. & Smiley, J. R. (2001). Herpes simplex virus triggers and then disarms a host antiviral response. J Virol 75, 750758.
Perelygin, A. A., Scherbik, S. V., Zhulin, I. B., Stockman, B. M., Li, I. & Brinton, M. A. (2002). Positional cloning of the murine flavivirus resistance gene. Proc Natl Acad Sci U S A 99, 93229327.
Prosniak, M., Hooper, D. C., Dietzschold, B. & Koprowski, H. (2001). Effect of rabies virus infection on gene expression in mouse brain. Proc Natl Acad Sci U S A 98, 27582763.
Rebouillat, D. & Hovanessian, A. G. (1999). The human 2', 5'-oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties. J Interferon Res 19, 295308.[CrossRef]
Reindl, M., Knipping, G., Wicher, I., Dilitz, E., Egg, R., Deisenhammer, F. & Berger, T. (2001). Increased intrathecal production of apolipoprotein D in multiple sclerosis. J Neuroimmunol 119, 327332.[CrossRef][Medline]
Renne, R., Barry, C., Dittmer, D., Compitello, N., Brown, P. O. & Ganem, D. (2001). Modulation of cellular and viral gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus. J Virol 75, 458468.
Samuel, C. E. (2001). Antiviral actions of interferons. Clin Microbiol Rev 14, 778809.
Samuel, C. E. (2002). Host genetic variability and West Nile virus susceptibility. Proc Natl Acad Sci U S A 99, 1155511557.
Smith, J. B. & Herschman, H. R. (1996). The glucocorticoid attenuated response genes GARG-16, GARG-39, and GARG-49/IRG2 encode inducible proteins containing multiple tetratricopeptide repeat domains. Arch Biochem Biophys 330, 290300.[CrossRef][Medline]
Su, H. L., Liao, C. L. & Lin, Y. L. (2002). Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response. J Virol 76, 41624171.
Thomas, E. A., Dean, B., Pavey, G. & Sutcliffe, J. G. (2001a). Increased CNS levels of apolipoprotein D in schizophrenic and bipolar subjects: implications for the pathophysiology of psychiatric disorders. Proc Natl Acad Sci U S A 98, 40664071.
Thomas, E. A., Sautkulis, L. N., Criado, J. R., Games, D. & Sutcliffe, G. J. (2001b). Apolipoprotein D mRNA expression is elevated in PDAPP transgenic mice. J Neurochem 79, 10591064.[CrossRef][Medline]
Tzamarias, D. & Struhl, K. (1995). Distinct TPR motifs of Cyc8 are involved in recruiting the Cyc8-Tup1 corepressor complex to differentially regulated promoters. Genes Dev 9, 821831.[Abstract]
Wynn, T. A., Nicolet, C. M. & Paulnock, D. M. (1991). Identification and characterization of a new gene family induced during macrophage activation. J Immunol 147, 43844392.
Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. (1998). Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays. Proc Natl Acad Sci U S A 95, 1447014475.
Received 12 September 2002;
accepted 11 February 2003.