Models and Mechanisms of Bornavirus Pathogenesis Martin Schwemmle1,*, W

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Drug Discovery Today: Disease Mechanisms Vol. 1, No. 2 2004

Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA

DRUG DISCOVERY Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA TODAY DISEASE MECHANISMS Infectious diseases

Models and mechanisms of Bornavirus pathogenesis Martin Schwemmle1,*, W. Ian Lipkin2,* 1Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany 2Jerome L. and Dawn Greene Infectious Disease Laboratory, Departments of Epidemiology, Neurology and Pathology, Mailman School of Public Health and College of Physicians and Surgeons, Columbia University, 722 West 168th Street, New York, NY 10032, USA

Borna Disease Virus (BDV) causes neurologic disease Section Editor: in a wide variety of warm-blooded animals. Although Anne Moscona – Department of Pediatrics, Mount Sinai human infection has not been conclusively demon- School of Medicine, One Gustave L. Levy Place, Box 1198, New York, NY 10029, USA strated, some surveys suggest that BDV or a related agent might infect humans and be implicated in mental Borna disease virus is a fascinating agent that causes persistent infection in the central nervous system of mammals, and there are tantalizing illness. Recent progress in tissue culture and animal suggestions that Borna is an etiological agent in a number of human neuropsychiatric disorders. This review highlights what is known about models reveals insights into the molecular biology of the viral life cycle and the role of both immune factors and specific viral this unique virus and unveils mechanisms by which factors in pathogenesis. Lipkin is internationally recognized for his work on immune and microbial factors in neurological and neuropsychiatric persistent, non-cytolytic viral infection can cause beha- diseases. In 1988, Lipkin and Oldstone made the seminal observation vioral disorders. that viral infection early in life can cause behavioral and neurotransmitter disturbances without obvious evidence of brain infection or injury. This finding that cryptic infection can influence brain Introduction function has been increasingly recognized as important in pathogenesis Borna Disease Virus (BDV) is an enveloped virus with a non- of neuropsychiatric diseases. Lipkin is at the forefront of Borna virus segmented, negative strand RNA genome. It includes six open research, and currently heads an international multi-center program to reading frames encoding the nucleoprotein (N), p10 (X), use novel methods to assess the role of Borna disease virus in human neuropsychiatric diseases. phosphoprotein (P), matrix protein (M), glycoprotein (G) and the polymerase (L). BDV is neurotropic, targets limbic structures, and causes persistent infection for the lifespan of observed in schizophrenia, mood disorders and autism the host. Natural infection is reported only in horses and through specific effects on neuronal plasticity and cellular sheep; however, experimental infections are described in a signaling pathways. This review highlights recent advances in wide variety of vertebrates and, dependent on the age and BDV research. immune status of the host, might present as florid immune- mediated disease or subtle behavioral alterations without overt inflammation. Rats are the best-characterized animal Main body text models of BDV pathogenesis. Whereas immunocompetent Molecular biology of Borna disease virus adults have meningoencephalitis, neonates have distur- The replication cycle of BDV is illustrated in Fig. 1. BDV bances in learning, mood and behavior reminiscent of those initiates infection (attachment) via binding of its envelope glycoprotein (G) to unidentified cellular receptor (s). After endocytosis the viral ribonucleoprotein complex (RNP) is *Corresponding authors: (M.Schwemmle) [email protected]; (Ian Lipkin) [email protected] released from the vesicles as a result of a pH-dependent

L is regulated by splicing of two introns. Whereas intron 1 is Glossary located within the M-ORF, intron 2 is found within the ORFs IFN: interferons are cytokines that induce multiple biological effects on for G and L [3]. After synthesis, N, P, and X enter the nucleus target cells, including anti-viral, anti-proliferative, and immunomodula- due to the presence of nuclear localization sequences (NLS). tory activities. Reverse genetic system: plasmid-based system to recover negative- The subcellular distribution of M is cytosolic and associated strand RNA polymerase activity or viruses entirely from cDNA. with cellular membranes. G is post-translationally modiﬁed by N-glycosylation and undergoes post-translational cleavage by the cellular protease furin; the resulting two protein frag- fusion. The RNP is composed of at least the RNA genome, L, P ments reach the cell surface to participate in the budding and N [1]. Recent crystallographic studies indicate that N process. This process involves the coordinated assembly of forms tetramers in the absence of viral RNA [2]. However, it is the viral RNPs into viral particles by M and the cleavage conceivable that the viral genome is wrapped around con- products of G. The mechanism by which RNP are exported secutive monomers. Replication and transcription of the viral from nucleus remains to be determined but might be genome occurs in the nucleus. Four major subgenomic RNAs mediated by N, as this protein contains a nuclear export are transcribed from three transcription units. Whereas N is sequence (NES) [3]. Budding occurs at the plasma membrane, encoded from a monocistronic transcript, all other proteins although intracellular budding from cytoplasmic membranes are derived from multicistronic RNAs. Expression of M, G and has been described. Remarkably, release of BDV particles from

Figure 1. BDV life cycle. After attachment and endocytosis of viral particles, the viral ribonucleoprotein complex (RNP), composed of the viral genome and associated BDV proteins, is released into the cytoplasm as a result of a pH-dependent fusion. Following nuclear import of the viral RNP, replication and transcription results in the continuous supply of the viral genome copies (anti-genomes) that serves again as a template to generate several copies of the viral genome, and spliced and unspliced RNA transcripts that code for 6 viral proteins. Of these proteins, N, P, X and L enter the nucleus to participate in the transcription and replication process. G and M remain in the cytoplasm, where G is post-translationally cleaved by a cellular protease. The two cleaved products participate with M at the plasma membrane in the budding process whereby RNPs are incorporated into viral particles.

212 www.drugdiscoverytoday.com Vol. 1, No. 2 2004 Drug Discovery Today: Disease Mechanisms | Infectious diseases the infected cell is a rare event. Tropism in the brain for or astrocytes are resistant to disease, presumably because of hippocampus and other limbic structures is probably deter- immunological tolerance [5]. Notably, the virus failed to mined at least in part by the distribution of PKC epsilon, the replicate in transgene-expressing granular and pyramidal cellular kinase required for phosphorylation of the BDV P. neurons of the hippocampus (preferred sites of replication) Functional analysis of the BDV polymerase complex has (Fig. 2), a finding consistent with the observation that stoi- been limited. Recently two laboratories established func- chiometry of RNP complex components is crucial to functional polymerase assays based on artificial minigenomes tionality. [1,4]. Early studies confirmed that N, P and L represent the Behavioral disturbances are different in adult and neona- active functional polymerase complex, revealed that X has a tally infected rats. Adult rats have a biphasic disorder linked negative regulatory function, and indicated that stoichiome- to abnormalities in dopamine neurotransmitter function. In try is critical to function. Rapid progress in establishment of the acute phase, coinciding with monocyte infiltration into reverse genetic systems (Glossary) seems imminent; these will the brain, rats have exaggerated startle responses and hyper- undoubtedly reveal insights into pathogenesis as well the activity. In the chronic phase, as infiltrates recede, rats show molecular biology of BDV (Outstanding issues). stereotyped motor behaviors. Neonatally infected rats have cerebellar and hippocampal dysgenesis, a wide range of subtle Animal models physiologic and neurobehavioral disturbances including The classic presentation in natural and experimental infec- runting, abnormal taste preferences, altered circadian tion is Borna disease (BD), an immune-mediated potentially rhythms, learning deficits, impaired play, and abnormalities fatal meningoencephaltis. However, the outcome of BD is in serotonin and glutamate systems. BDV is not cytotoxic; variable with age, host genetic factors and neurovirulence of thus, the pathogenesis of neurodevelopmental damage in the the isolate. Studies in rats and mice indicate that immuno- absence of immunopathology remains unclear. However, pathology in classical disease is mediated chiefly by N pro- clues can come from in vitro studies wherein infection impacts tein-specific CD8+ T cells. Interestingly, acute disease can signaling pathways that could be critical for establishment and recede despite persistence of replicating virus. This apparent maintenance of neural architecture (Outstanding issues). BDV tolerance is associated with a shift from a Th1 to Th2 blocks extracellular signal-regulated kinase (ERK)-mediated weighted immune response but the basis for this shift is neuronal differentiation of PC12 cells in response to the nerve unexplained. Transgenic mice expressing N in either neurons growth factor (NGF). Activated (phoshorylated) ERK fails to

Figure 2. Neurons of transgenic mice expressing BDV-N are resistant to BDV infection. BDV-N transgenic mice (a and c) and non-transgenic mice (b and d) were infected with BDV. Several weeks post-infection expression of the N (transgene) and P (marker for infection) were analyzed in the hippocampus of these animals. N-transgenic hippocampal neurons were resistant to infection. Arrows indicate absence of P expression.

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Figure 3. Potential mechanisms of neural dysfunction. (a) In persistently BDV-infected rat pheochromocytoma (PC12) cells, the NGF-induced signaling cascade is inhibited at the level of activated (phosphorylated) ERK. By binding to its tyrosine kinase receptor TrkA, NGF induces via the small GTPase Ras the serial activation of kinases by phosphorylation, including Raf, MEK and ERK. Phosphorylation and nuclear translocation of the latter kinase results in the activation of transcription factors including Elk1 and CREB. In BDV-infected PC12 cells the translocation of activated ERK is inhibited. Reduced activation of transcription factors might impair neuronal differentiation. The viral gene products implicated are unknown. (b) BDV P might impair cell motility, growth of cell processes and transcriptional activation through interference with the function of HMGB1. HMGB1 induces intracellular and extracellular signaling events by acting either directly in the nucleus or by binding to the RAGE receptor. The latter event results in the induction of the Ras-Raf-MEK-ERK pathway and the activation of the transcription factors NF-kb and SP1. It also activates the small GTPase Cdc42 and Rac1 that regulate cell motility. BDV P might bind to HMGB1 in the nucleus or the cytoplasm. (c) BDV interferes with the transition from G2 to M phase by binding to the inactivated Cdk1–Cyclin B1 complex. Progression from G2 to M phase involves the activation of Cdk1–Cyclin B1 complex by removal of two phosphate residues (black encircled P) of Cdk1 by the phosphatase CDC25 and subsequent nuclear translocation of the complex. Ubiquitin-dependent cyclin B1 degradation results in inactive Cdk1. BDV-N blocks transition from inactive to active Cdk1/cyclinB1 complex.

translocate efficiently into the nucleus, resulting in an activity (Fig. 3b). Lastly, BDV N might interfere with the cell impaired phosphorylation of transcription factors, including cycle by preventing the activation of the cyclin-dependent Elk-1 and cAMP response element binding protein (CREB; Fig. kinase 1 (Cdk1)/Cyclin B1 complex in G2 phase (Fig. 3c) [10]. 3a) [6]. In hippocampal neurons, BDV also blocks the brain- In concert, these findings indicate several mechanisms by derived neurotropic factor (BDNF), nerve growth factor (NGF) which BDV might cause disease in the absence of an extrinsic pathway at a site upstream of ERK [7].Recentdatasuggestthat inflammatory response. Whether specific viral gene products BDV interferes with neurite outgrowth and cell motility by can be implicated remains to be determined. Behavioral interacting with the high-mobility group box protein 1 abnormalities are observed in transgenic mice expressing (HMGB1 or amphoterin) [8]. HMGB1 facilitates the assembly BDV P in glial cells [11]; these include enhanced intermate of site-specific DNA binding proteins, such as p53, to their aggressiveness, hyperactivity and spatial memory deficits. cognate binding sites within chromatin. In addition to these However, the loss of neurons in hippocampus and cerebellum, intracellular functions, HMGB1 has several extracellular func- which is the sine qua non of neonatal BDV infection, is not tions, including the activation of the RAGE (receptor for found in BDV P transgenic mice. Additionally, neurons rather advanced glycation end products) receptor that can lead to than glial cells are the primary site of BDV replication. None- neurite outgrowth and migration of cells. Experimental data theless, although the significance of this model for neonatal suggest that BDV P binds to HMGB1 in the nucleus as well BDV infection is unclear it provides an intriguing new tool as in the cytoplasm [8,9], thus interfering with HMGB1- with which to study behavioral disorders in the absence of induced RAGE signaling and p53-mediated transcriptional virus infection.

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Table 1. Key therapies Therapy Stage of development Advantages and/or disadvantages Refs. Neutralizing antibodies Cell culture [12] IFN Cell culture Efficient/not effective in some cell types [16,17] 1B6TM Cell culture [20] Ribavarin Cell culture/animal Moderate efficiency [13–15] Ara-C Cell culture/animal Efficient/cytotoxic side effects [18] 20-FdC Cell culture Reduced toxicicty compared to Ara-C [12]

Abbreviations: IFN, interferon; Ara-C, 1-b-d-arabinofuranosylcytosine; 20FDC, 20-ﬂuoro-20-deoxycytidin; 1B6TM, 1-O-benzyl-6-O-titryl-a-d-mannosepyranoside.

Borna disease virus and human disease Links The epidemiology and consequences of human infection have been controversial since 1985 when the first serologic Griffin, D.E. (2003) Immune responses to RNA-virus infections of the CNS. Nat. Rev. Immunol. 3, 493–502 studies emerged implicating BDV in bipolar disorder (Out- Johnson, R.T. (2003) Emerging viral infections of the nervous system. standing issues). Most reports implicating BDV and human J. Neurovirol. 9, 140–147 disease have focused on neuropsychiatric disorders includ- Yolken, R.H. and Torrey, E.F. (1995) Viruses, schizophrenia, and ing unipolar depression, bipolar disorder, or schizophrenia; bipolar disorder. Clin. Microbiol. Rev. 8, 131–145 Billich, C. et al. (2002) High-avidity human serum antibodies however, BDV has also been linked to chronic fatigue recognizing linear epitopes of Borna disease virus proteins. Biol. syndrome, AIDS encephalopathy, multiple sclerosis, motor Psychiatry 51, 979–987 neuron disease, and brain tumors (glioblastoma multi- Bode, L. and Ludwig, H. (2003) Borna disease virus infection, a human mental–health risk. Clin. Microbiol. Rev. 16, 534–545 forme). Isolation of infectious virus is only rarely reported. Carbone, K.M. et al. (2002) Borna disease virus (BDV)-induced model Infection is more frequently diagnosed by serology or PCR of autism: application to vaccine safety test design. Mol. Psychiatry 7 analyses of peripheral blood mononuclear cells or tissues. (Suppl. 2), S36–S37 Schwemmle, M. et al. (1997) Borna disease virus P-protein is Serologic studies including cell-based immunofluorescence, phosphorylated by protein kinase C e and casein Kinase II. J. Biol. ELISA, western blot and immune complex assays have Chem. 272, 21818–21823 revealed antibodies to BDV in subjects with selected neuropsychiatric disorders with a prevalence rate of 0–93% versus 0–15% of normal controls. The differences in the dies to the BDV G protein inhibit virus fusion and spread in prevalence rates presumably reflect differences in specificity tissue culture [12]; whether, passive immunotherapy is ther- and sensitivity of the assays employed. However, because apeutic in vivo remains to be established. Cerebral spinal fluid there are no sera from humans confirmed to be infected it filtration is reported to improve clinical status of BDV has been difficult to establish an accepted method for infected humans with affective or schizophrenic psychosis. validating assays or ruling out false positive or negative The mechanism is likely to be alteration of levels of cytokines, reactions. Several groups have reported identification of antibodies, or other molecules with inflammatory or neu- BDV nucleic acids from blood or brain tissue by nested roactive properties rather than an anti-viral effect. One RT-PCR. Although sensitive, this method is prone to artifact research group has found amantadine to be effective reducing because of inadvertent introduction of template from virus load and ameliorating disease in infected horses with laboratory isolates or cross contamination of samples. In most viral systems, specific signatures readily facilitate determination of provenance; however, in BDV, similarities in sequence between putative new isolates and confirmed Related articles isolates cannot be used to exclude the former as artifacts. Planz, O. et al. (2002) Human Borna disease virus infection. In Borna The epidemiology of BDV and its significance in human Disease Virus and its role in Neurobehavioral Disease (Carbone, K.M., ed.), disease awaits multicenter studies wherein standardized pp. 179–225, ASM Press Hornig, M. et al. (2003) Borna disease virus. J. Neurovirol. 9, 259–273 methods are employed for clinical diagnosis and blinded Lipkin, W.I. and Hornig, M. (2004) Psychotropic viruses. Curr. Opin. laboratory assessment of infection. Microbiol. 7, 420–425 Stitz, L. et al. (2002) The immunopathogenesis of Borna disease virus infection. Front Biosci. 7, d541–d555 Therapy of BDV infection Staeheli, P. et al. (2000) Epidemiology of Borna disease virus. J. Gen. Virol. Both immunomodulatory and chemical therapies are pro- 81, 2123–2135 posed to treat BDV infection (Table 1). Monoclonal antibo-

www.drugdiscoverytoday.com 215 Drug Discovery Today: Disease Mechanisms | Infectious diseases Vol. 1, No. 2 2004 encephalitis and in infected humans with neuropsychiatric Outstanding issues disorders. However, others have found no anti-viral efficacy in vitro or in animal models and have suggested that clinical What is the epidemiology of BDV? What are its implications for humans? improvement in neuropsychiatric disease reflects activity at What are the mechanisms by which BDV establishes persistence in the N-methyl-D-aspartate receptor. As noted earlier, there is the central nervous system? no consensus concerning human infection; thus, indivi- How does BDV interfere with signaling pathways to lead to the duals who responded to cerebral spinal fluid filtration or selective loss of neuronal cells? amantadine might or might not have been infected with References BDV. Ribavirin is effective in vitro in reducing BDV replica- 1 Schneider, U. et al. (2003) Active borna disease virus polymerase complex tion [13,14] and in vivo in reducing morbidity and mortality requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral X [15]. Interestingly, in vivo efficacy appears to be linked protein. J. Virol. 77, 11781–11789 primarily to immunomodulatory effects on microglia rather 2 Rudolph, M.G. et al. (2003) Crystal structure of the borna disease virus nucleoprotein. Structure (Camb.) 11, 1219–1226 than anti-viral activity [15]. BDV replication is sensitive to a/ 3 Tomonaga, K. et al. (2002) Molecular and cellular biology of Borna disease b and g interferons (IFN, Glossary); efficacy in vivo is not virus infection. Microbes Infect. 4, 491–500 4 Perez, M. et al. (2003) A reverse genetics system for Borna disease virus. J. reported [16,17].1-b-D-arabinofuranosylcytosine (Ara-C), Gen. Virol. 84, 3099–3104 inhibits BDV replication in cultured cells and inhibits 5 Rauer, M. et al. (2004) Transgenic mice expressing the nucleoprotein of viral replication and improves clinical outcome in infected Borna disease virus in either neurons or astrocytes: decreased susceptibility rats [18]. The mechanism for this therapeutic response is to homotypic infection and disease. J. Virol. 78, 3621–3632 6 Hans, A. et al. (2001) Borna disease virus persistent infection activates proposed to be a specificeffectontheBDVLpolymerase. mitogen-activated protein kinase and blocks neuronal differentiation of Ara-C is a DNA polymerase inhibitor, and has no effect PC12 cells. J. Biol. Chem. 276, 7258–7265 on polymerases of influenza or measles virus; nonetheless, 7 Hans, A. et al. (2004) Persistent, noncytolytic infection of neurons by the inhibitory effect of Ara-C in BDV infection appears to Borna disease virus interferes with ERK 1/2 signaling and abrogates BDNF-induced synaptogenesis. FASEB J. 18, 863–865 be inhibition of the viral polymerase. A related cytosine 8 Kamitani, W. et al. (2001) Borna disease virus phosphoprotein binds a nucleoside, 20-FdC, shows similar anti-viral activity neurite outgrowth factor, amphoterin/HMG-1. J. Virol. 75, 8742–8751 in vitro and might be preferred because of reduced cytotoxi- 9 Zhang, G. et al. (2003) Borna disease virus phosphoprotein represses p53- mediated transcriptional activity by interference with HMGB1. J. Virol. 77, city [19]. 12243–12251 10 Planz, O. et al. (2003) Borna disease virus nucleoprotein interacts with the Conclusion CDC2-cyclin B1 complex. J. Virol. 77, 11186–11192 11 Kamitani, W. et al. (2003) Glial expression of Borna disease virus BDV is the prototype of a new family and genus within the phosphoprotein induces behavioral and neurological abnormalities in non-segmented RNA viruses. Its broad host range, and beha- transgenic mice. Proc. Natl. Acad. Sci. USA 100, 8969–8974 vioral consequences in natural and experimental infection 12 Bajramovic, J.J. et al. (2003) Borna disease virus glycoprotein is required suggest that BDV has potential to cause human neuropsy- for viral dissemination in neurons. J. Virol. 77, 12222–12231 13 Jordan, I. et al. (1999) Inhibition of Borna disease virus replication by chiatric disease. Although the epidemiology and clinical ribavirin. J. Virol. 73, 7903–7906 significance of BDV are not yet clarified, studies in animal 14 Mizutani, T. et al. (1998) Inhibition of Borna disease virus replication by models have allowed dissection of pathways by which viruses ribavirin in persistently infected cells. Arch. Virol. 143, 2039–2044 15 Solbrig, M.V. et al. (2002) Neuroprotection and reduced proliferation of can persist and cause brain dysfunction. Recent advances in microglia in ribavirin-treated bornavirus-infected rats. Antimicrob. Agents BDV reverse genetics and cellular biology afford new oppor- Chemother. 46, 2287–2291 tunities for functional analyses that will provide insights into 16 Hallensleben, W. and Staeheli, P. (1999) Inhibition of Borna disease virus multiplication by interferon: cell line differences in susceptibility. Arch. this unique agent and mechanisms of pathogenesis in human Virol. 144, 1209–1216 diseases ranging from immune-mediated meningoencepha- 17 Friedl, G. et al. (2004) Borna disease virus multiplication in mouse litis to neurodevelopmental disorders. organotypic slice cultures is site-specifically inhibited by gamma interferon but not by interleukin-12. J. Virol. 78, 1212–1218 18 Bajramovic, J.J. et al. (2002) 1-Beta-D-arabinofuranosylcytosine inhibits Acknowledgements borna disease virus replication and spread. J. Virol. 76, 6268–6276 We thank Daniel Gonzalez-Dunia and Gerald Radziwill 19 Bajramovic, J.J. et al. (2004) 20-fluoro-20-deoxycytidine inhibits Borna for discussion of BDV effects on signaling pathways. The disease virus replication and spread. Antimicrob. Agents Chemother. 48, 1422–1425 figure showing the neurons of transgenic mice expressing 20 Stoyloff, R. et al. (1998) Neutralization of borna disease virus depends BDV-N was provided by Ju¨rgen Hausmann and Mathias upon terminal carbohydrate residues (alpha-D-man, beta-D-GlcNAc) of Rauer. glycoproteins gp17 and gp94. Intervirology 41, 135–140

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