Theiler's murine encephalomyelitis virus (TMEV) subgroup strain-specific infection in neural and non-neural cell lines.

GDVII subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) are highly virulent and produce acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. In contrast, DA subgroup strains are less virulent and establish a persistent central nervous system infection which results in demyelinating disease. We previously reported a subgroup-specific infection in a macrophage-like cell line, J774-1 cells; i.e., GDVII strain does not replicate in J774-1 cells, whereas the DA strain actively replicates in these cells. In addition, this subgroup-specific virus growth is shown to be related to the presence of L* protein, a 17 kDa protein translated out-of-frame of the viral polyprotein from an AUG located 13 nucleotides downstream from the polyprotein's AUG. The present paper demonstrated that this subgroup-specific infection is observed in murine monocyte/macrophage lineage cell lines, but not in other murine cell lines including neural cells. An RNase protection assay also suggested that L* protein-related virus growth is regulated at the step of viral RNA replication. As macrophages are reported to be the major cell harboring virus during the chronic demyelinating stage, the activity of L* protein with respect to virus growth in macrophages may be a key factor in clarifying the mechanism(s) of TMEV persistence, which is probably a trigger to spinal cord demyelination.     

Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.     

Chimeric cDNA studies of Theiler's murine encephalomyelitis virus neurovirulence.

Strain GDVII and other members of the GDVII subgroup of Theiler's murine encephalomyelitis virus are highly neurovirulent and rapidly fatal, while strain DA and other members of the TO subgroup produce a chronic, demyelinating disease. GDVII/DA chimeric cDNA studies suggest that a major neurovirulence determinant is within the GDVII 1B through 1D capsid protein coding region, although the additional presence of upstream GDVII sequences, including the 5' untranslated region, contributes to full neurovirulence. Our studies indicate that there are limitations in precisely delineating neurovirulence determinants with chimeric cDNAs between evolutionarily diverged viruses, such as GDVII and DA.     

Theiler's murine encephalomyelitis virus as a vaccine candidate for immunotherapy

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.     

A subgenomic segment of Theiler's murine encephalomyelitis virus RNA causes demyelination

The DA strain of Theiler's murine encephalomyelitis virus (TMEV) causes a persistent central nervous system (CNS) infection of mice with a restricted virus gene expression and induces an inflammatory demyelinating disease that is thought to be immune mediated and a model of multiple sclerosis (MS). The relative contribution of virus vis-à-vis the immune system in the pathogenesis of DA-induced white matter disease remains unclear, as is also true in MS. To clarify the pathogenesis of DA-induced demyelination, we used Cre/loxP technology to generate a transgenic mouse that has tamoxifen (Tm)-inducible expression of a subgenomic segment of DA RNA in oligodendrocytes and Schwann cells. Tm-treated young transgenic mice developed progressive weakness leading to death, with abnormalities of oligodendrocytes and Schwann cells and demyelination, but without inflammation, demonstrating that DA virus can play a direct pathogenic role in demyelination. Tm treatment of mice at a later age resulted in milder disease, with evidence of peripheral nerve remyelination and focal fur depigmentation; surviving weak mice had persistent expression of the recombined transgene in the CNS, suggesting that the DA subgenomic segment can cause cellular dysfunction but not death, possibly similar to the situation seen during DA virus persistence. These studies demonstrate that DA RNA or a DA protein(s) is toxic to myelin-synthesizing cells. This Cre/loxP transgenic system allows for spatially and temporally controlled expression of the viral transgene and is valuable for clarifying nonimmune (and immune) mechanisms of demyelination induced by TMEV as well as other viruses.     

Apoptotic and antiapoptotic activity of L protein of Theiler's murine encephalomyelitis virus

Cellular apoptosis induced by viral genes can play a critical role in determining virulence as well as viral persistence. This form of cell death has been of interest with respect to Theiler's murine encephalomyelitis virus (TMEV) because the GDVII strain and members of the GDVII subgroup are highly neurovirulent, while the DA strain and members of the TO subgroup induce a chronic progressive inflammatory demyelination with persistence of the virus in the central nervous system. The TMEV L protein has been identified as important in the pathogenesis of Theiler's virus-induced demyelinating disease (TMEV-IDD). We now show that DA L is apoptotic following transfection of L expression constructs or following DA virus infection of HeLa cells; the apoptotic activity depends on the presence of the serine/threonine domain of L, especially a serine at amino acid 57. In contrast, GDVII L has little apoptotic activity following transfection of L expression constructs in HeLa cells and is antiapoptotic following GDVII infection of HeLa cells. Of note, both DA and GDVII L cleave caspase-3 in BHK-21 cells, although neither implements the full apoptotic machinery in this cell type as manifested by the induction of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. The differences in apoptotic activities of DA and GDVII L in varied cell types may play an important role in TMEV subgroup-specific disease phenotypes.     

Theiler's murine encephalomyelitis virus subgroup strain-specific infection in a murine macrophage-like cell line.

We compared infection of a murine macrophage-like cell line, J774-1, with two Theiler's murine encephalomyelitis virus subgroup strains. The GDVII strain, which is highly virulent and produces acute polioencephalomyelitis in mice, did not actively replicate in J774-1 cells, although there was a significant inhibition in cellular protein synthesis. In contrast, the DA strain, which is less virulent and causes demyelination with a persistent virus infection, productively infected J774-1 cells; however, there was less virus produced than in BHK-21 cells, and there was little if any cellular protein shutoff. These in vitro data may provide some explanation for the biological activities that are observed between both subgroup strains.     

The leader peptide of Theiler's murine encephalomyelitis virus is a zinc-binding protein.

The leader (L) peptide is located in the amino-terminal part of the polyprotein of members of the Cardiovirus (which includes Theiler's murine encephalomyelitis virus) and Aphthovirus genera of picornaviruses. Although the function of L is unknown, strain DA of Theiler's murine encephalomyelitis virus with a mutation of L produces a cell-specific restricted infection. We now report that the DA L peptide is a metalloprotein and that zinc binds to a Cys-His motif that is conserved among cardioviruses.     

Neurotropic viral infections leading to epilepsy: focus on Theiler's murine encephalomyelitis virus

Neurotropic viruses cause viral encephalitis and are associated with the development of seizures/epilepsy. The first infection-driven animal model for epilepsy, the Theiler's murine encephalomyelitis virus-induced seizure model is described herein. Intracerebral infection of C57BL/6 mice with Theiler's murine encephalomyelitis virus induces acute seizures from which the animals recover. However, once the virus is cleared, a significant portion of the animals that experienced acute seizures later develop epilepsy. Components of the innate immune response to viral infection, including IL-6 and complement component 3, have been implicated in the development of acute seizures. Multiple mechanisms, including neuronal cell destruction and cytokine activation, play a role in the development of acute seizures. Future studies targeting the innate immune response will lead to new therapies for seizures/epilepsy.     

L* protein of Theiler's murine encephalomyelitis virus is required for virus growth in a murine macrophage-like cell line

We sought to confirm the importance of L* protein for growth of Theiler's murine encephalomyelitis virus (TMEV) in a macrophage-like cell line, J774-1. The protein is out of frame with the polyprotein and synthesized in DA but not GDVII subgroup strains of TMEV. A recombinant virus, DANCL*/GD, which substitutes the DA 5' noncoding and L* coding regions for the corresponding regions of GDVII and synthesizes L* protein, grew with little restriction in J774-1 cells. In contrast, another recombinant virus, DANCL*-1/GD, which has an ACG rather than an AUG as the starting codon of L* protein at nucleotide 1079, resulting in no synthesis of L* protein, did not grow well. No significant difference between the rates of adsorption to J774-1 cells of these viruses was observed. RNase protection assay demonstrated that DANCL*/GD viral RNA significantly increased, whereas only a minimal increase was observed for DANCL*-1/GD. The present study suggests that L* protein is required for virus growth in macrophages.     

Expression of the vesicular stomatitis virus membrane glycoprotein gene in Bacillus subtilis

Abstract The molecularly cloned gene encoding the vesicular stomatitis virus (VSV) membrane glycoprotein G was modified and joined to a Bacillus subtilis secretion vector constructed from the plasmid pUB110 and containing the promoter and signal sequence regions of the α-amylase (a secretory protein) gene from Bacillus amyloliquefaciens . The regions encoding the NH₂-terminal signal peptide and the COOH-terminal hydrophobic transmembrane domains of the VSV gene were deleted to facilitate the secretion of the G protein in soluble form. The truncated G protein was found to be expressed in B. subtilis . The expression level was low, probably due to rapid proteolytic degradation of the protein and, contrary to what was expected, almost all of the protein remained cell-associated.     

Differentiation of M1 myeloid precursor cells into macrophages results in binding and infection by Theiler's murine encephalomyelitis virus and apoptosis

Infection of susceptible mouse strains with BeAn, a less virulent strain of Theiler's murine encephalomyelitis virus (TMEV), results in immune system-mediated demyelinating lesions in the central nervous system (CNS) similar to those in multiple sclerosis. Since macrophages appear to carry the major detectable antigen burden in vivo, and purification of sufficient cell numbers from the CNS for detailed analysis is difficult, macrophage-like cell lines provide an accessible system with which to study virus-macrophage interactions. The myeloid precursor cell line M1 differentiates in response to cytokines and expresses many characteristics of tissue macrophages. Incubation of TMEV with undifferentiated M1 cells produced neither infection nor apoptosis, whereas differentiated M1 (M1-D) cells developed a restricted virus infection and changes indicative of apoptosis. Virus binding and RNA replication as well as cellular production of alpha/beta interferons increased with differentiation. Although the amount of infectious virus was highly restricted, BeAn-infected M1-D cells synthesized and appropriately processed virus capsid proteins at levels comparable to those for permissive BHK-21 cells. Analysis of Bcl-2 protein family expression in undifferentiated and differentiated cells suggests that susceptibility of M1-D cells to apoptosis may be controlled, in part, by expression of the proapoptotic alpha isoform of Bax and Bak. These data suggest that macrophage differentiation plays a role in susceptibility to TMEV infection and apoptosis.     

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.     

Theiler's murine encephalomyelitis virus leader protein amino acid residue 57 regulates subgroup-specific virus growth on BHK-21 cells

Strains of Theiler's murine encephalomyelitis virus (TMEV) are divided into two subgroups, TO and GDVII. TMEV strains show subgroup-specific virus growth and cell tropism and induce subgroup-specific diseases. Using site-directed mutagenesis, we demonstrated that the amino acid at position 57 of the leader protein (L(57)), which is located at the most N-terminal part of the polyprotein, regulates subgroup-specific virus growth on BHK-21 cells. Further study suggested that L(57) may regulate viral RNA encapsidation, although it does not affect the synthesis of viral proteins or the assembly of viral intermediates.