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.

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.

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.

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.

An early, abundant cytotoxic T-lymphocyte response against Theiler's virus is critical for preventing viral persistence.

In genetically susceptible strains of mice, the DA strain of Theiler's virus, a picornavirus, causes a persistent infection of the white matter of the spinal cord associated with chronic demyelination. In resistant strains, on the other hand, the infection is cleared within 1 to 2 weeks. In this article, we show that Theiler's virus induces a rapid and abundant cytotoxic T lymphocyte (CTL) response in resistant C57BL/6 mice, while the response remains low throughout infection in susceptible SJL/J mice. This difference can be referred to a higher number of virus-specific CTL precursors in C57BL/6 mice. These observations indicate that the efficient induction of virus-specific CTL precursors is critical for avoiding the establishment of a persistent picornaviral infection.

Role of macrophages during Theiler's virus infection.

Theiler's virus, a murine picornavirus, causes a persistent infection of the central nervous system with chronic inflammation and primary demyelination. We examined the nature of infected cells at different times postinoculation (p.i.) with a combined immunocytochemistry-in situ hybridization assay. The virus was found in the gray matter of the brain, mostly in neurons, during the first week p.i. During the following weeks, the virus was present in the spinal cord, first in the gray and white matter, then exclusively in the white matter. Approximately 10% of infected cells were astrocytes at any time during the study. Infected oligodendrocytes were first noticed on day 14 p.i. and amounted to approximately 6% of infected cells. The number of infected macrophages increased with time and reached a plateau by day 21 p.i., when at least 45% of infected cells were macrophages. The role of blood-borne macrophages during infection was studied by depleting them with mannosylated liposomes containing dichloromethylene diphosphonate. The virus did not persist in the majority of the mice treated with liposomes. These mice showed only minimal mononuclear cell infiltration and no demyelination.

Infection of choroid plexus cells by human T cell leukaemia virus type I.

Very little is known about the factors that determine the outcome of infection by human T cell leukaemia virus type I (HTLV-I) and the neurotropism of this virus is still a controversial point. In transgenic mice, the HTLV-I LTR is active mainly in the central nervous system (CNS), in parenchyma as well as in ependymal and choroid plexus cells. The latter are of particular interest and could represent the way of entry of the virus into the CNS. In this study we show that primary cultures of sheep choroid plexus can be infected with HTLV-I, leading to characteristic multinucleated syncytial cells containing virus RNA and proteins. HTLV-I p24 Gag protein was detected in the culture medium and the presence of virus particles was observed by electron microscopy 40 days after infection. At this time post-infection HTLV-I could be transmitted to human cord blood cells.

In vivo administration of interleukin-2 protects susceptible mice from Theiler's virus persistence.

In vivo administration of interleukin-2 (IL-2)-secreting tumor cells results in complete protection against persistent infection by Theiler's murine encephalomyelitis virus (TMEV) in susceptible DBA/2 mice. The IL-2-mediated protection was found to depend on the inoculum size as well as the timing of IL-2 administration. IL-2-treated and TMEV-infected mice displayed a three- to fourfold relative increase in virus-specific cytotoxic T-lymphocyte (CTL) precursors. Thus, we postulate that the persistence of TMEV infection in susceptible mice reflects limited numbers of relevant CTL precursors and their time course of induction and activation.

Analysis of the expression directed by two HTLV-I promoters in transgenic mice.

We generated several lines of mice transgenic for the lacZ reporter gene under the control of an HTLV-I LTR. Two different LTR were used; one was isolated from a case of Adult T-cell Leukemia (ATL), the other from a case of Tropical Spastic Paraparesis (TSP/HAM). These LTR differed at 18 nucleotide positions. The pattern of expression of the transgene, studied at the RNA level by RT-PCR, was the same regardless of the origin of the promoter. The beta-galactosidase activity was detected primarily in the central nervous system, in the parenchyma, the choroid plexus and the ependymal cells along the ventricles. In parenchyma, double labelling experiments showed that the cells expressing beta-galactosidase were neurons. These results show that choroid plexus cells and ependymal cells, as well as some neurons, are permissive for the activity of the HTLV-I promoter. The origin of the LTR had not influence on the pattern of expression of the reporter gene.

A search for human T-cell leukemia virus type I in the lesions of patients with tropical spastic paraparesis and polymyositis.

We searched for the presence of human T-cell leukemia virus type I (HTLV-I) sequences in central nervous system and muscle lesions of 3 patients with tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) and 3 patients with HTLV-I-associated polymyositis. Proviral DNA coding for the Tax protein was found by polymerase chain reaction amplification in DNA extracted from lesions of every patient with TSP/HAM or HTLV-I-associated polymyositis. In contrast, viral RNA was found occasionally by in situ hybridization in muscle lesions of some patients with polymyositis, but was never found in central nervous system lesions of TSP/HAM patients.

The gene coding for interferon-gamma is linked to the D12S335 and D12S313 microsatellites and to the MDM2 gene.

Interferon-gamma is a cytokine with multiple effects. It interferes with the replication of several viruses and plays a key role in the regulation of immune responses. Therefore, the gene coding for interferon-gamma could be implicated in the susceptibility of humans to several diseases. We have localized this gene close to the D12S335 and D12S313 microsatellites on both the physical and the genetic maps of the human genome. We also physically mapped this gene close to the MDM2 locus on chromosome band 12q15. Finally, we describe the organization of the Ifg, Myf-6, Mdm1, and Mdm2 loci on mouse chromosome 10, in a region syntenic to human chromosome band 12q15.

Isolation from human brain of six previously unreported cDNAs related to the reverse transcriptase of human endogenous retroviruses.

cDNAs prepared from total RNA extracted from plaques of multiple sclerosis were amplified by the polymerase chain reaction. The 11-bp degenerate primers used were derived from conserved sequences of reverse transcriptase. Amplified cDNAs were fractionated according to size by electrophoresis in polyacrylamide gels under denaturing conditions. cDNAs of the proper size were cloned, grouped according to the sequence of their insert by differential hybridization, and sequenced. Six cDNAs were isolated and found to belong to new members of two groups of human endogenous retroviruses: the group related to ERV9 and that related to HERVK10 and HUMMTV. These sequences were expressed in all human organs tested, including normal white matter of brain. The approach described in this article is a powerful tool with which to isolate new members of the reverse transcriptase gene family.

Comparative analysis of HTLV-I promoter activities reveals no disease-linked pattern of expression.

To investigate the possibility of an association between the type of pathology caused by HTLV-I and the activity of its promoter, we compared the levels of transcription obtained with six LTRs isolated from patients with two different HTLV-I-related diseases: ATL and TSP/HAM. The patients came from different geographical endemic areas. The LTR region was amplified by polymerase chain reaction (PCR) from the DNA of uncultured peripheral blood lymphocytes, and directly cloned upstream of the luciferase reporter gene. Constructs were tested by a transient transfection assay in a variety of cell lines. Although the activities of these LTRs were statistically different in some of the cell lines tested, no correlation could be demonstrated between the promoter activity and the nature of the disease. Thus, the data suggest that the LTR is not a major determinant of the nature of the disease associated with the infection by HTLV-I.

Functional analysis of two long terminal repeats from the HTLV-I retrovirus.

Human T-cell leukemia virus type I (HTLV-I) is associated with a large spectrum of clinical manifestations in man. Viral and host factors are probably involved in determining the consequences of infection. Although most of the genome of HTLV-I appears remarkably stable, considerable variation is observed in the long terminal repeat (LTR) which harbors the promoter region. So far, no correlation between specific mutations and pathogenesis has been found, and the current opinion is that sequence variations reflect the geographical origin of the isolate more than the associated pathology. To assess whether the mutations observed between two HTLV-I LTRs were functionally significant, two LTRs, which differ by ten mutations, were coupled to the highly sensitive eukaryotic luciferase-encoding reporter gene, luc, and tested by transfection in a variety of cell lines. Marked differences in promoter activity were observed in some of the cells tested, whereas in other both LTRs were equally active. This result demonstrates that the minor differences observed between two HTLV-I LTRs can affect the activity level of the promoter in some cellular environments, a result which could point to the LTR as one determinant of HTLV-I cell tropism in vivo.

Theiler's virus replication in brain macrophages cultured in vitro.

Infection of the mouse with Theiler's virus is one of the best animal models for the study of multiple sclerosis, a chronic demyelinating disease of the human central nervous system. The identification of the virus target cell(s) is fundamental to an understanding of the viral persistence as well as the inflammation and demyelination observed in the chronic phase of the disease. This paper reports that a small fraction of brain macrophages grown in vitro can be efficiently infected with Theiler's virus without significant cytolytic effect. Viral replication as well as continuous production of infectivity were observed in these cultures.

Tissue expression pattern directed in transgenic mice by the LTR of an HTLV-I provirus isolated from a case of tropical spastic paraparesis.

Human T lymphotropic virus type I (HTLV-I) causes adult T cell leukemia/lymphoma and a chronic neurological disease called either tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy. The different outcomes of this infection could be due to both host and viral factors and it has been proposed that genetic differences could make some HTLV-I strains neurotropic. In this paper, we examined the pattern of tissue-specific expression determined by a long terminal repeat (LTR) obtained from a case of TSP. We constructed transgenic mice in which this LTR controlled the expression of the nlslacZ reporter gene. We observed that in three independent lines of transgenic mice, the reporter gene was expressed predominantly in the central nervous system (CNS), in choroid plexus, and in cells of the hippocampus and cerebellum. Our observations indicate the existence of CNS cells permissive for the expression of HTLV-I and which may be of importance in the pathogenesis of TSP.

Theiler's virus infection induces a specific cytotoxic T lymphocyte response.

Theiler's virus, a murine picornavirus, persists in the central nervous system of susceptible mouse strains and causes chronic inflammation and primary demyelination. One of the current hypotheses is that demyelination is, at least in part, mediated by virus-specific cytotoxic T lymphocytes (CTL). However, it is generally assumed that picornaviruses do not induce CTL. In point of fact, their existence has only been demonstrated for Coxsackievirus B-3. To determine whether Theiler's virus induces a CTL response, we generated a murine mastocytoma cell line stably transfected with the coding region of the genome of Theiler's virus strain DA. Using these cells as targets we showed that infected DBA/2 mice, a susceptible strain, produce cytotoxic T lymphocytes. The cytotoxic activity was Theiler's-virus specific. It was for the most part mediated by CD8+ T lymphocytes and H-2 restricted. This is the first demonstration that a specific CTL response is generated during Theiler's virus infection.

Analysis of retroviral sequences in the spinal form of multiple sclerosis.

The polymerase chain reaction was used, in a blind study, to look for retroviral sequences in DNA extracted from the peripheral blood mononuclear cells of 11 patients with the spinal form of multiple sclerosis (MS). Control subjects consisted of 7 patients with other neurological diseases and 5 healthy blood donors. Three sets of oligonucleotides were used. They could detect all known human oncoretroviruses, lentiviruses, or spumaretroviruses. The primers recognized conserved sequences in the long terminal repeats of the proviral DNA. Control experiments showed that the primers crossreacted within the human immunodeficiency virus or human T-cell lymphotropic virus group and that they provided the expected level of sensitivity. Therefore the assay could have detected not only known human retroviruses but also new related members. In spite of this, no retroviral sequences were detected in either the MS or the control specimen.