Interleukin 22 is a candidate gene for Tmevp3, a locus controlling Theiler's virus-induced neurological diseases.

After intracerebral inoculation, Theiler's virus induces in its natural host, the mouse, an acute encephalomyelitis followed, in susceptible animals, by chronic inflammation and primary demyelination. Susceptibility to demyelination among strains of laboratory mice is explained by the capacity of the immune system to control viral load during persistence. Also, differences of susceptibility to viral load between the susceptible SJL strain and the resistant B10.S strain are mainly due to two loci, Tmevp2 and Tmevp3, located close to the Ifng locus on chromosome 10. In this article, we show that the Tmevp3 locus controls both mortality during the acute encephalomyelitis and viral load during persistence. Most probably, two genes located in the Tmevp3 interval control these two different phenotypes with efficiencies that depend on the age of the mouse at inoculation. Il22, a member of the IL-10 cytokine family, is a candidate gene for the control of mortality during the acute encephalomyelitis.

Homology between a 173-kb region from mouse chromosome 10, telomeric to the Ifng locus, and human chromosome 12q15.

We sequenced a 173-kb region of mouse chromosome 10, telomeric to the Ifng locus, and compared it with the human homologous sequence located on chromosome 12q15 using various sequence analysis programs. This region has a low density of genes: one gene was detected in the mouse and the human sequences and a second gene was detected only in the human sequence. The mouse gene and its human orthologue, which are expressed in the immune system at a low level, produce a noncoding mRNA. Nonexpressed sequences show a higher degree of conservation than exons in this genomic region. At least three of these conserved sequences are also conserved in a third mammalian species (sheep or cow).

Viral load increases in SJL/J mice persistently infected by Theiler's virus after inactivation of the beta(2)m gene.

We show that inactivating the beta(2)m gene increases the viral load of SJL/J mice persistently infected by Theiler's virus. Together with previous results, this shows that the characteristics of Tmevp1, a locus which controls the amount of viral RNA that persists in the central nervous system, are those of an H-2 class I gene.

Disruption of differentiated functions during viral infection in vivo. V. Mapping of a locus involved in susceptibility of mice to growth hormone deficiency due to persistent lymphocytic choriomeningitis virus infection.

Lymphocytic choriomeningitis virus (LCMV) Armstrong strain selectively and persistently infects the majority of growth hormone (GH) producing cells in the anterior lobe of pituitary glands of C3H/St mice but negligibly infects GH producing cells of BALB/WEHI mice (Oldstone et al., Virology 142, 175--182, 1985; Oldstone et al., Science 218, 1125--1127, 1982). Although infected GH cells remain free of structural damage, disrupted initiation of GH transcription (Klavinskis and Oldstone, J. Gen. Virol. 68, 1867--1873, 1989; Valsamakis et al., Virology 156, 214--220, 1987) occurs with a resultant decrease in the synthesis of GH, leading to a failure of growth and development (Oldstone et al., Science 218, 1125--1127, 1982). Microsatellite mapping of DNA obtained from 101 individual C3H/St x BALB/WEHI F1 x F1 mice shows that the growth failure correlates with host genes linked (P value 0.0008) on chromosome 17 just outside of the H-2D MHC site between D17 Mit24 and D17 Mit51, a distance of 2.5 cM. The genetic mapping done here excludes alpha-dystroglycan (alpha-DG), a known receptor for LCMV (Cao et al., Science 282, 2079--2081, 1998) in pathogenesis of GH disease, as alpha-DG is encoded in the mouse by a gene residing on chromosome 9 (Yotsumoto et al., Hum. Mol. Genet. 5, 1259--1267, 1996).

Borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of PC12 cells.

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.

H-2D(b-/-) mice are susceptible to persistent infection by Theiler's virus.

H-2(b) mice are resistant to persistent infection of the central nervous system by Theiler's virus. They clear the infection 7 to 10 days after intracranial inoculation. Resistance maps to the H-2D gene and not to the H-2K gene and is associated with a potent antiviral cytotoxic T-lymphocyte (CTL) response. We used H-2(b) mice in which the H-2D or the H-2K gene had been inactivated to dissect the respective roles of these genes in resistance. We report that H-2D(-/-) but not H-2K(-/-) mice were susceptible to persistent infection. Furthermore, whereas H-2K(-/-) mice mounted a vigorous virus-specific CTL response, similar to that of control C57BL/6 mice, the CTL response of H-2D(-/-) mice was nil or minimal. Using target cells transfected with the H-2D(b) or the H-2K(b) gene, we showed that the H-2K-restricted CTL response against the virus was minimal in H-2D(-/-) mice. These results demonstrate that the H-2D(b) and H-2K(b) genes play nonredundant roles in the resistance to this persistent infection.

Cytokines in genetic susceptibility to multiple sclerosis: a candidate gene approach. French Multiple Sclerosis Genetics Group.

The immune system is involved in the pathophysiology of multiple sclerosis (MS) but the initiating antigen(s) is not yet identified. Since cytokines control both the intensity and the quality of the immune response they may be relevant candidates for the genetic susceptibility to MS. To analyze the contribution of type 1 and type 2 cytokine and cytokine receptor genes in the genetic susceptibility to MS, we have examined, in 116 French MS sibpairs, whether there is significant linkage between MS and 15 cytokine or cytokine receptor genes using 31 highly polymorphic genetic markers. The data were analyzed using the maximum likelihood score and the transmission disequilibrium approaches. None of the candidate genes tested was significantly linked to MS on the whole population. However, after stratification of the analysis on the basis of sharing (or not) of the HLA-DRB1*1501 allele, indication of linkage was found for the IL2-RB gene. These findings suggest that the IL2-RB locus contributes to the genetic susceptibility in a subgroup of MS patients.

Viral load and a locus on chromosome 11 affect the late clinical disease caused by Theiler's virus.

Theiler's virus causes a persistent infection and a demyelinating disease of mice which is a model for multiple sclerosis. Susceptibility to viral persistence maps to several loci, including the interferon gamma locus. Inactivating the gene coding for the interferon gamma receptor makes 129/Sv mice susceptible to persistent infection and clinical disease, whereas inactivating the interferon gamma gene makes C57BL/6 mice susceptible to persistent infection but not to clinical disease. This difference in phenotype is due to the difference in genetic background. Clinical disease depends on high viral load and Tmevd5, a locus on chromosome 11. These results have consequences for the identification of viruses which might be implicated in multiple sclerosis.

The Th1/Th2 balance does not account for the difference of susceptibility of mouse strains to Theiler's virus persistent infection.

Theiler's virus causes a persistent infection with demyelination that is studied as a model for multiple sclerosis. Inbred strains of mice differ in their susceptibility to viral persistence due to both H-2 and non-H-2 genes. A locus with a major effect on persistence has been mapped on chromosome 10, close to the Ifng locus, using a cross between susceptible SJL/J and resistant B10.S mice. We now confirm the existence of this locus using two lines of congenic mice bearing the B10.S Ifng locus on an SJL/J background, and we describe a deletion in the promoter of the Ifng gene of the SJL/J mouse. We studied the expression of IFN-gamma, IL-2, IL-10, and IL-12 in the brains of SJL/J mice, B10.S mice, and the two lines of congenic mice during the first 2 wk following inoculation. We found a greater expression of IFN-gamma and IL-2 mRNA in the brains of B10.S mice compared with those of SJL/J mice. Also, the ratio of IL-12 to IL-10 mRNA levels was higher in B10.S mice. However, the cytokine profiles were the same for the two lines of resistant congenic mice and for susceptible SJL/J mice. Therefore, the difference of Th1/Th2 balance between the B10.S and SJL/J mice is not due to the Ifng locus and does not account for the difference of susceptibility of these mice to persistent infection.

The GDVII strain of Theiler's virus spreads via axonal transport.

Following intracerebral inoculation, the DA strain of Theiler's virus sequentially infects neurons in the gray matter and glial cells in the white matter of the spinal cord. It persists in the latter throughout the life of the animal. Several observations suggest that the virus spreads from the gray to the white matter by axonal transport. In contrast, the neurovirulent GDVII strain causes a fatal encephalitis with lytic infection of neurons. It does not infect the white matter of the spinal cord efficiently and does not persist in survivors. The inability of this virus to infect the white matter could be due to a defect in axonal transport. Using footpad inoculations, we showed that the GDVII strain is, in fact, transported in axons. Transport was prevented by sectioning the sciatic nerve. The kinetics of transport and experiments using colchicine suggested that the virus uses microtubule-associated fast axonal transport. Our results show that a cardiovirus can spread by fast axonal transport and suggest that the inability of the GDVII strain to infect the white matter is not due to a defect in axonal transport.

Two loci, Tmevp2 and Tmevp3, located on the telomeric region of chromosome 10, control the persistence of Theiler's virus in the central nervous system of mice.

Theiler's virus persistently infects the white matter of the spinal cord in susceptible strains of mice. This infection is associated with inflammation and primary demyelination and is studied as a model of multiple sclerosis. The H-2D gene is the major gene controlling viral persistence. However, the SJL/J strain is more susceptible than predicted by its H-2(s) haplotype. An (SJL/J x B10. S)F1 x B10.S backcross was analyzed, and one quantitative trait locus (QTL) was located in the telomeric region of chromosome 10 close to the Ifng locus. Another one was tentatively mapped to the telomeric region of chromosome 18, close to the Mbp locus. We now report the study of 14 congenic lines that carry different segments of these two chromosomes. Although the presence of a QTL on chromosome 18 was not confirmed, two loci controlling viral persistence were identified on chromosome 10 and named Tmevp2 and Tmevp3. Furthermore, the Ifng gene was excluded from the regions containing Tmevp2 and Tmevp3. Analysis of the mode of inheritance of Tmevp2 and Tmevp3 identified an effect of sex, with males being more infected than females.

Distinct attenuation phenotypes caused by mutations in the translational starting window of Theiler's murine encephalomyelitis virus.

Upon initiation of translation of picornavirus RNA, the ribosome is believed to bind the internal ribosome entry site of the template and then to form a productive complex with a downstream RNA segment, the starting window. The presence or absence of an AUG triplet within the starting window of the RNA of Theiler's murine encephalomyelitis virus (a picornavirus) is known to modulate its neurovirulence. In this study, mutants of this virus in which the starting windows, lying upstream of the viral polyprotein reading frame, had AUGs with different nonoptimal contexts were engineered. Upon intracerebral inoculation of mice, the mutants proved to be partially attenuated, as judged by a significant increase in the dose causing paralysis in 50% of the animals (PD50). Mutants with similar PD50s might differ from one another by eliciting either a severe, fatal tetraplegy or only mild, recoverable neurologic lesions. Some of the mutants triggered a chronic inflammatory reaction in the white matter of the spinal cord in the absence of detectable viral RNA or antigen. Thus, point mutations changing the context of an AUG within the starting window outside the polyprotein reading frame may differently affect the morbidity and mortality caused by a viral infection and may result in distinct attenuation phenotypes.

An attenuated variant of the GDVII strain of Theiler's virus does not persist and does not infect the white matter of the central nervous system.

The DA strain of Theiler's virus causes a persistent and demyelinating infection of the white matter of spinal cord, whereas the GDVII strain causes a fatal gray-matter encephalomyelitis. Studies with recombinant viruses showed that this difference in phenotype is controlled mainly by the capsid. However, conflicting results regarding the existence of determinants of persistence in the capsid of the GDVII strain have been published. Here we show that a GDVII virus whose neurovirulence has been attenuated by an insertion in the 5' noncoding region does not persist in the central nervous systems of mice. Furthermore, this virus infects the gray matter efficiently, but not the white matter. These results confirm the absence of determinants of persistence in the GDVII capsid. They suggest that the DA capsid controls persistence by allowing the virus to infect cells in the white matter of the spinal cord.

Interaction of Theiler's virus with intermediate filaments of infected cells.

Theiler's murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler's virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.

Genetics of susceptibility to Theiler's virus infection.

Theiler's virus is a picornavirus of mouse which causes an acute encephalomyelitis followed by a persistent infection of the white matter resulting in chronic inflammation and demyelination. This disease has been studied as a model for multiple sclerosis. Inbred strains of mice are either resistant--they clear the infection after the acute encephalomyelitis--or susceptible to persistent infection and demyelination. Susceptibility is a polygenic trait which has been analyzed using methods of association with "candidate" genes, and linkage analysis after a complete genome scan. The H-2Db gene is responsible for an efficient CTL response which makes some strains resistant. Non H-2 genes responsible for the susceptibility of other strains have been mapped by linkage analysis to the lfng and, possibly, the Mbp loci. The analysis of a set of congenic mice ruled out the possibility that the relevant gene codes for interferon gamma, and showed that the region around lfng probably contains two susceptibility genes. The analysis of mutant mice showed further that the Mbp gene, which codes for the myelin basic protein, has a major effect on viral persistence.

Viruses and multiple sclerosis.

Animal models illustrate how viruses and host genetic factors may interact to cause immune-mediated demyelination. Similar mechanisms may take place in at least some forms of multiple sclerosis, a disease that is histopathologically heterogeneous. No 'multiple sclerosis virus' has been found yet, although recent data on human herpesvirus-6 antigens in multiple sclerosis brain warrant further investigation. Multiple sclerosis associated retrovirus, a recently described retroviral sequence isolated from multiple sclerosis material, is a member of the endogenous retrovirus-9 family. The association between the expression of this virus associated retrovirus and multiple sclerosis is only tentative.

The neurovirulence of the DA and GDVII strains of Theiler's virus correlates with their ability To infect cultured neurons.

The strains of Theiler's murine encephalomyelitis virus, a picornavirus, are divided into two groups according to their neurovirulence after intracerebral inoculation. The highly virulent GDVII strain causes an acute, fatal encephalomyelitis, whereas the DA strain causes a mild encephalomyelitis followed by a chronic inflammatory demyelinating disease associated with viral persistence. Studies with recombinant viruses showed that the capsid plays the major role in determining these phenotypes. However, the molecular basis for the effect of the capsid on neurovirulence is still unknown. In this paper, we describe a large difference in the patterns of infection of primary neuron cultures by the GDVII and DA strains. Close to 90% of the neurons were infected 12 h after inoculation with the GDVII strain, and the cytopathic effect was complete 24 h postinoculation. In contrast, with the DA strain, viral antigens were not detected in neurons until 24 h postinoculation. Infected neurons accounted for only 2% of the total number of neurons, even 6 days after inoculation. No cytopathic effect was visible, and the cultures could be kept for the same length of time as the noninfected controls. Because the neurovirulence of the GDVII strain has been mapped to the capsid, we examined the role of the capsid in this difference of phenotype. We showed, using recombinant viruses, that the capsid was indeed responsible for the pattern of infection observed in vitro, most likely through its role in viral entry. Thus, the levels of neurovirulence of the GDVII and DA strains correlate with their abilities to infect cultured neurons, and this ability is controlled by the capsid.

Molecular mechanism of tumorigenesis in mice transgenic for the human T cell leukemia virus Tax gene.

The infection by human T lymphotropic virus type I is associated with adult T cell leukemia and several inflammatory degenerative disorders, including tropical spastic paraparesis. To investigate the role of the Tax protein in the development of diseases linked to human T lymphotropic virus type I infection, we generated two lines of transgenic mice carrying the tax gene under the control of the viral promoter. The expression of the transgene was low in these mice and was restricted to the central nervous system and testis. Mice from both lines developed various types of tumors, including fibrosarcomas and adenocarcinomas. Tax was expressed at a high level in fibrosarcomas and in cell lines derived from these tumors. In tumor-derived cells, the expression of Tax led to an increased degradation of IkappaB alpha and IkappaB beta and caused stable nuclear translocation of nuclear factor-kappaB. This translocation was essential for cell proliferation, as shown by expressing a nondegradable form of IkappaBbeta in these cells. Therefore, Tax-induced cell transformation in mice correlates with the degradation of IkappaB alpha and IkappaB beta and with the constitutive activation of NF-kappaB.

Chromosome 14 contains determinants that regulate susceptibility to Theiler's virus-induced demyelination in the mouse.

Theiler's murine encephalomyelitis virus causes a chronic demyelinating disease in susceptible strains of mice that is similar to human multiple sclerosis. Several nonmajor histocompatibility complex-linked genes have been implicated as determinants of susceptibility or resistance to either demyelination or virus persistence. In this study, we used linkage analysis of major histocompatibility complex identical H-2d (DBA/2J x B10.D2) F2 intercross mice to identify loci associated with susceptibility to virus-induced demyelinating disease. In a 20-cM region on chromosome 14, we identified four markers, D14Mit54, D14Mit60, D14Mit61, and D14Mit90 that are significantly associated with demyelination. Because two peaks were identified, one near D14Mit54 and one near D14Mit90, it is possible that two loci in this region are involved in controlling demyelination.

Theiler's virus infection of perforin-deficient mice.

Theiler's virus, a murine picornavirus, infects the central nervous systems of C57BL/6 mice and is cleared after approximately 10 days by a process which requires CD8+ cytotoxic T cells. We used perforin-deficient C57BL/6 mice to test the role of this protein in viral clearance. Perforin-deficient mice died from viral encephalomyelitis between days 12 and 18 postinoculation. They had high levels of viral RNA in their central nervous systems until the time of death. In contrast, viral RNA had disappeared by day 11 postinoculation in wild-type C57BL/6 mice. Cytotoxic T cells can kill infected cells by two main mechanisms: the secretion of the pore-forming protein perforin or the interaction of the Fas ligand with the apoptosis-inducing Fas molecule on the target cell. Our results demonstrate that clearance of Theiler's virus from the central nervous system in C57BL/6 mice is perforin dependent.