Neutralizing monoclonal antibodies to Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are serologically related picornaviruses which cause both enteric and neurological disease in mice. The biological activities of TMEV vary between the two different TMEV subgroups (TO and GDVII) and with different passage histories of the same TMEV strain (e.g., mouse brain-passed versus tissue culture-passed DA strain of the TO subgroup). We raised neutralizing monoclonal antibodies (mAbs) against tissue culture-passed DA and GDVII strains of TMEV. We produced two mAbs against the DA strain which neutralized all members of the TO subgroup, but not the GDVII subgroup strains (GDVII and FA); these two DA mAbs reacted similarly with both mouse brain-passed DA and tissue culture-passed DA. Of six neutralizing GDVII mAbs, four reacted only to GDVII and FA, whereas two neutralized TO strains as well. These mAbs demonstrate the presence of TMEV group-specific as well as subgroup-specific neutralization and substantiate the division of TMEV into two distinct subgroups. On Western immunoblots one of the two DA mAbs reacted against isolated DA VP1, two GDVII mAbs (which were TMEV group specific) reacted against isolated GDVII VP1 and DA VP1, and the other DA mAb and four other GDVII mAbs required an intact virion conformation for reactivity. An analysis of the epitopes recognized by these mAbs may elucidate sites important in TMEV biological activities.     

Strains from both Theiler's virus subgroups encode a determinant for demyelination.

The GDVII strain and other members of the GDVII subgroup of Theiler's murine encephalomyelitis viruses (TMEV) cause an acute lethal neuronal infection in mice, whereas the DA strain and other members of the TO subgroup of TMEV cause a chronic demyelinating disease associated with a persistent virus infection. We used GDVII/DA chimeric infectious cDNAs to produce intratypic recombinant viruses in order to clarify reasons for the TMEV subgroup-specific difference in demyelinating activity. We found that both the GDVII and DA strains contain a genetic determinant(s) for demyelinating activity. No demyelination occurs following GDVII strain inoculation because this strain produces an early neuronal disease that kills mice before white matter disease and persistent infection can occur.     

Role of sialyloligosaccharide binding in Theiler's virus persistence.

Theiler's murine encephalomyelitis viruses (TMEVs) belong to the Picornaviridae family and are divided into two groups, typified by strain GDVII virus and members of the TO (Theiler's original) group. The highly virulent GDVII group causes acute encephalitis in mice, while the TO group is less virulent and causes a chronic demyelinating disease which is associated with viral persistence in mice. This persistent central nervous system infection with demyelination resembles multiple sclerosis (MS) in humans and has thus become an important model for studying MS. It has been shown that some of the determinants associated with viral persistence are located on the capsid proteins of the TO group. Structural comparisons of two persistent strains (BeAn and DA) and a highly virulent strain (GDVII) showed that the most significant structural variations between these two groups of viruses are located on the sites that may influence virus binding to cellular receptors. Most animal viruses attach to specific cellular receptors that, in part, determine host range and tissue tropism. In this study, atomic models of TMEV chimeras were built with the known structures of GDVII, BeAn, and DA viruses. Comparisons among the known GDVII, BeAn, and DA structures as well as the predicted models for the TMEV chimeras suggested that a gap on the capsid surface next to the putative receptor binding site, composed of residues from VP1 and VP2, may be important in determining viral persistence by influencing virus attachment to cellular receptors, such as sialyloligosaccharides. Our results showed that sialyllactose, the first three sugar molecules of common oligosaccharides on the surface of mammalian cells, inhibits virus binding to the host cell and infection with the persistent BeAn virus but not the nonpersistent GDVII and chimera 39 viruses.     

L* Protein of the DA Strain of Theiler’s Murine Encephalomyelitis Virus Is Important for Virus Growth in a Murine Macrophage-Like Cell Line

Strain GDVII and other members of the GDVII subgroup of Theiler’s murine encephalomyelitis virus (TMEV) are highly virulent and cause acute polioencephalomyelitis in mice. Neither viral persistence nor demyelination is demonstrated in the few surviving mice. On the other hand, strain DA and other members of the TO subgroup of TMEV are less virulent and establish a persistent infection in the spinal cord, which results in a demyelinating disease. We previously reported that GDVII does not actively replicate in a murine macrophage-like cell line, J774-1, whereas DA strain productively infects these cells (M. Obuchi, Y. Ohara, T. Takegami, T. Murayama, H. Takada, and H. Iizuka, J. Virol. 71:729–733, 1997). In the present study, we used recombinant viruses between these strains of the two subgroups to demonstrate that the DA L coding region of DA strain is important for virus growth in J774-1 cells. Additional experiments with a mutant virus indicate that L* protein, which is synthesized out of frame with the polyprotein from an additional alternative initiation codon in the L coding region of TO subgroup strains, is a key determinant responsible for the cell-type-specific restriction of virus growth. L* protein may play a critical role in the DA-induced restricted demyelinating infection by allowing growth in macrophages, a major site for virus persistence.     

Mutations that affect the tropism of DA and GDVII strains of Theiler's virus in vitro influence sialic acid binding and pathogenicity

Theiler's murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse. The different strains of TMEV are divided into two subgroups according to the pathology they provoke. The neurovirulent strains GDVII and FA induce an acute fatal encephalitis, while persistent strains, like DA and BeAn, cause a chronic demyelinating disease associated with viral persistence in the central nervous system. Different receptor usage was proposed to account for most of the phenotype difference between neurovirulent and persistent strains. Persistent but not neurovirulent strains were shown to bind sialic acid. We characterized DA and GDVII derivatives adapted to grow on CHO-K1 cells. Expression of glycosaminoglycans did not influence infection of CHO-K1 cells by parental and adapted viruses. Mutations resulting from adaptation of DA and GDVII to CHO-K1 cells notably mapped to the well-characterized VP1 CD and VP2 EF loops of the capsid. Adaptation of the DA virus to CHO-K1 cells correlated with decreased sialic acid usage for entry. In contrast, adaptation of the GDVII virus to CHO-K1 cells correlated with the appearance of a weak sialic acid usage for entry. The sialic acid binding capacity of the GDVII variant resulted from a single amino acid mutation (VP1-51, Asn-->Ser) located out of the sialic acid binding region defined for virus DA. Mutations affecting tropism in vitro and sialic acid binding dramatically affected the persistence and neurovirulence of the 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 (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.     

Theiler's murine encephalomyelitis virus--characterization of newly isolated viruses from Japanese mouse colonies

The hemagglutinating-inhibition (HI) test was used to detect antibodies for Theiler's murine encephalomyelitis virus (TMEV), and the virus was isolated from sero-positive mice derived from colonies in Japan. HI antibody was detected in conventional mice (38.7%; 137/354) at titers ranging from 1:8 to 1:512, but it not in SPF mice (0/90). To isolate the virus, weanling mice inoculated intracerebrally with samples obtained from sero-positive mice were sacrificed and 10% brain homogenates were subcultured. New isolates designated as YOC and AB strains were obtained, and their physicochemical and biological properties were characterized. The results indicated that the new isolates were similar to Theiler's original (TO) strain according to the following observations of persistent paralysis of the hind limbs, resistance to ether treatment, a particles size of 10 approximately 50 nm in diameter, stability at pH 3, a density of 1.35 g/cm3 and three major and one minor viral proteins, (VPO; 38 Kd, VP 1; 33 Kd, VP2; 32Kd, VP3; 25 Kd). Immunoblotting analysis also showed that VP 2 of YOC and encephalomyocarditis virus of the Cardiovirus group, reacted strongly with the antisera against the viruses as well as with the GDVII strain. These results suggest that TMEV infection does exist in conventional mouse colonies in Japan, and that these viruses resemble the TO strain of TMEV.     

Analysis of Cellular Mutants Resistant to Theiler’s Virus Infection: Differential Infection of L929 Cells by Persistent and Neurovirulent Strains

Theiler's murine encephalomyelitis virus (TMEV) is a natural pathogen of the mouse and belongs to the Picornaviridae family. TMEV strains are divided into two subgroups on the basis of their pathogenicity. The first group contains two neurovirulent strains, FA and GDVII, which cause a rapid fatal encephalitis. The second group includes persistent strains, like DA and BeAn, which produce a biphasic neurological disease in susceptible mice. Persistence of these viruses in the white matter of the spinal cord leads to chronic inflammatory demyelination. L929 cells, which are susceptible to TMEV infection, were subjected to physicochemical mutagenesis. Cellular clones that became resistant to TMEV infection were selected by viral infection. Three such mutants resistant to strain GDVII were characterized to determine the step of the virus cycle that was inhibited. The mutation present in one of these mutant cell lines inhibited, by more than 1,000-fold, the entry of strain GDVII but hardly decreased infection by strain DA. In the two other cellular mutants, replication of the viral genome was slowed down. Interestingly, one of these mutant cell lines resisted infection by both the persistent and neurovirulent strains while the second cell line resisted infection by strain GDVII but remained susceptible to the persistent virus. These results show that although they have 95% identity at the amino acid sequence level, neurovirulent and persistent viruses use partly distinct pathways for both entry into cells and genome replication.     

A Determinant for Central Nervous System Persistence Localized in the Capsid of Theiler’s Murine Encephalomyelitis Virus by Using Recombinant Viruses

The demyelinating process in Theiler’s murine encephalomyelitis virus (TMEV) infection in mice requires virus persistence in the central nervous system. Using recombinant TMEV assembled between the virulent GDVII and less virulent BeAn virus cDNAs, we now provide additional evidence supporting the localization of a persistence determinant to the leader P1 (capsid) sequences. Further, recombinant viruses in which BeAn sequences progressively replaced those of GDVII within the capsid starting at the leader NH2 terminus suggest that a conformational determinant requiring homologous sequences in both the VP2 puff and VP1 loop regions, which are in close contact on the virion surface, might underlie persistence.     

Antibodies to the trypsin cleavage peptide VP8 neutralize rotavirus by inhibiting binding of virions to target cells in culture.

Two distinct patterns of neutralization were identified by comparing the neutralization curves of monoclonal antibodies (MAbs) directed at the two surface proteins, VP4 and VP7, of rhesus rotavirus. VP7-specific MAbs were able to neutralize virus efficiently, and slight increases in antibody concentration resulted in a sharp decline in infectivity. On the other hand, MAbs to VP4 proved much less efficient at neutralizing rhesus rotavirus, and the fraction of infectious virus decreased gradually throughout a wide range of antibody concentrations. MAbs directed at VP8*, the smaller trypsin cleavage fragment of VP4, were shown to efficiently prevent binding of radiolabeled virions to MA104 cell monolayers, to an extent and at concentrations comparable to those required for neutralization of infectivity. Conversely, MAbs recognizing VP7 or the larger VP4 trypsin cleavage product, VP5*, showed little or no inhibitory effect on virus binding to cells. All MAbs studied were able to neutralize rotavirus that was already bound to the surface of cells. The MAbs directed at VP8*, but not those recognizing VP5* or VP7, were shown to mediate release of radiolabeled virus from the surface of the cells. With MAbs directed at VP7, papain digestion of virus-bound antibody molecules led to an almost complete recovery of infectivity. Neutralization could be fully restored by incubation of virus-Fab complexes with anti-mouse immunoglobulin G antiserum. Neutralization with MAbs directed at VP8* proved insensitive to digestion with papain as well as to the addition of anti-immunoglobulin antibodies.     

Sialylation of the host receptor may modulate entry of demyelinating persistent Theiler's virus

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus of the Cardiovirus genus. Certain strains of TMEV may cause a chronic demyelinating disease, which is very similar to multiple sclerosis in humans, associated with a persistent viral infection in the mouse central nervous system (CNS). Other strains of TMEV only cause an acute infection without persistence in the CNS. It has been shown that sialic acid is a receptor moiety only for the persistent TMEV strains and not for the nonpersistent strains. We report the effect of sialylation on cell surface on entry and the complex structure of DA virus, a persistent TMEV, and the receptor moiety mimic, sialyllactose, refined to a resolution of 3.0 A. The ligand binds to a pocket on the viral surface, composed mainly of the amino acid residues from capsid protein VP2 puff B, in the vicinity of the VP1 loop and VP3 C terminus. The interaction of the receptor moiety with the persistent DA strain provides new understanding for the demyelinating persistent infection in the mouse CNS by TMEV.     

A Protein Critical for a Theiler’s Virus-Induced Immune System-Mediated Demyelinating Disease Has a Cell Type-Specific Antiapoptotic Effect and a Key Role in Virus Persistence

TO subgroup strains of Theiler’s murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection and demyelinating disease in mice. This disease serves as an experimental model of multiple sclerosis (MS) because the two diseases have similar inflammatory white matter pathologies and because the immune system appears to mediate demyelination in both processes. We previously reported (H. H. Chen, W. P. Wong, L. Zhang, P. L. Ward, and R. P. Roos, Nat. Med. 1:927–931, 1995) that TO subgroup strains use an alternative initiation codon (in addition to the AUG used to synthesize the picornavirus polyprotein from one long open reading frame) to translate L*, a novel protein that is out of frame with the polyprotein and which plays a key role in the demyelinating disease. We now demonstrate that L* has antiapoptotic activity in macrophage cells and is critical for virus persistence. The antiapoptotic action of L* as well as the differential translation of L* and virion capsid proteins may foster virus persistence in macrophages and interfere with virus clearance. The regulation of apoptotic activity in inflammatory cells may be important in the pathogenesis of TMEV-induced demyelinating disease as well as MS.     

Determinants of persistence and demyelination of the DA strain of Theiler''s virus are found only in the VP1 gene.

The DA strain of Theiler's virus persists in the central nervous systems of mice and causes chronic inflammation and demyelination. The GDVII strain, on the other hand, causes an acute encephalitis that kills the host in a matter of days. We constructed a series of recombinants between two infectious cDNA clones of the genomes of DA and GDVII viruses. Analysis of the phenotypes of the recombinant viruses yielded the following results. (i) Determinants of persistence and demyelination are found only in the VP1 capsid protein of DA virus. (ii) Whereas the VP1 capsid protein of DA virus is able to fully attenuate the neurovirulence of GDVII virus and to allow the chimeric virus to persist and demyelinate, the VP1 capsid protein of GDVII virus is unable to render DA virus neurovirulent. (iii) The mere attenuation of the neurovirulence of GDVII virus does not allow it to persist and demyelinate.     

Differential usage of carbohydrate co-receptors influences cellular tropism of Theiler's murine encephalomyelitis virus infection of the central nervous system

Theiler's murine encephalomyelitis viruses (TMEV) are ubiquitous pathogens of mice, producing either rapidly fatal encephalitis (high-neurovirulence strains) or persistent central nervous system infection and inflammatory demyelination (low-neurovirulence strains). Although a protein entry receptor has not yet been identified, carbohydrate co-receptors that effect docking and concentration of the virus on the cell surface are known for both TMEV neurovirulence groups. Low-neurovirulence TMEV use α2,3-linked N-acetylneuramic acid (sialic acid) on an N-linked glycoprotein, whereas high-neurovirulence TMEV use the proteoglycan heparan sulfate (HS) as a co-receptor. While the binding of low-neurovirulence TMEV to sialic acid can be inhibited completely, only a third of the binding of high-neurovirulence TMEV to HS is inhibitable, suggesting that high-neurovirulence strains use another co-receptor or bind directly to the putative protein entry receptor. Four amino acids on the surface (VP2 puff B) of low-neurovirulence strains make contact with sialic acid through non-covalent hydrogen bonds. Since these virus residues are conserved in all TMEV strains, the capsid conformation of this region is probably responsible for sialic acid binding. A persistence determinant that maps within the virus coat using recombinant TMEV is also conformational in nature. Low-neurovirulence virus variants that do not bind to sialic acid fail to persist in the central nervous system of mice, indicating a role for sialic acid binding in TMEV persistence. Analysis of high-neurovirulence variants that do not bind HS demonstrates that HS co-receptor usage influences neuronal tropism in brain, whereas, the HS co-receptor use is not required for the infection of spinal cord anterior horn cells associated with poliomyelitis.     

Transgenic expression of Theiler's murine encephalomyelitis virus genes in H-2(b) mice inhibits resistance to virus-induced demyelination

We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2(b)) congenic mice expressing three independent contiguous coding regions of the Theiler's murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8(+) T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8(+) T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.     

Different Subcellular Localization of Theiler's Murine Encephalomyelitis Virus Leader Proteins of GDVII and DA Strains in BHK-21 Cells

The highly virulent GDVII strain of Theiler''s murine encephalomyelitis virus causes acute and fatal encephalomyelitis, whereas the DA strain causes mild encephalomyelitis followed by a chronic inflammatory demyelinating disease with virus persistence. The differences in the amino acid sequences of the leader protein (L) of the DA and GDVII strains are greater than those for any other viral protein. We examined the subcellular distribution of DA L and GDVII L tagged with the FLAG epitope in BHK-21 cells. Wild-type GDVII L was localized predominantly in the cytoplasm, whereas wild-type DA L showed a nucleocytoplasmic distribution. A series of the L mutant experiments demonstrated that the zinc finger domain, acidic domain, and C-terminal region of L were necessary for the nuclear accumulation of DA L. A GDVII L mutant with a deletion of the serine/threonine (S/T)-rich domain showed a nucleocytoplasmic distribution, in contrast to the predominant cytoplasmic distribution of wild-type GDVII L. A chimeric DA/GDVII L, D/G, which encodes the N region of DA L including the zinc finger domain and acidic domain, followed by the GDVII L sequence including the S/T-rich domain, was distributed exclusively throughout the cytoplasm but not in the nucleus, as observed with wild-type GDVII L. Another chimeric L, G/D (which is the converse of the D/G construct), accumulated in the nucleus as well as the cytoplasm, as was observed for wild-type DA L. The findings suggest that the differential distribution of DA L and GDVII L is determined primarily by the S/T-rich domain. The S/T-rich domain may be important for the viral activity through the regulation of the subcellular distribution of L.Theiler''s murine encephalomyelitis virus (TMEV) belongs to the genus Cardiovirus of the family Picornaviridae, and its strains are divided into two subgroups on the basis of their different biological activities. The neurovirulent strains, such as GDVII and FA, produce acute and fatal encephalomyelitis in mice. The persistent strains, such as TO, DA, BeAn, etc., induce mild and nonfatal encephalomyelitis, followed by a chronic demyelinating disease with virus persistence in the spinal cords of mice. This late demyelinating disease is thought to be an excellent experimental model for the human demyelinating disease multiple sclerosis (MS) (5, 17, 20).The TMEV genome is a single-stranded RNA molecule and translated as a long precursor polyprotein to yield 12 viral proteins by autoproteolytic cleavage (23). Two subgroup strains of TMEV have a sequence identity of approximately 95% at the amino acid level. The amino acid sequences of the proteins encoded by the P1, P2, and P3 regions of both strains are highly conserved and show 94, 96, and 98% identity, respectively. The genome has another coding region, designated the leader (L), at the most amino-terminal location of the precursor polyprotein. The L coding region encodes 76 amino acids (aa) and shows a low sequence identity of only 85% to the above-described three regions (16, 19, 22). Therefore, L has the greatest difference in amino acid sequence among any of the viral proteins and may play an important role in subgroup-specific biological activities of TMEV. In this study, we have investigated the subcellular localization of the L proteins of GDVII and DA strains and characterized the functional domains involved in the differential distribution between DA L and GDVII L in BHK-21 cells by a series of deletion mutant and chimeric construct experiments.     

Pathogenesis of early and late disease in mice infected with Theiler''s virus, using intratypic recombinant GDVII/DA viruses.

Intratypic recombinant Theiler's viruses prepared between GDVII and DA strains were used to identify genomic sequences important in neurovirulence, virus persistence, and demyelination and to clarify the mechanisms involved in disease induction. The coding region between 1B and 2C of the highly virulent GDVII strain contains a determinant partly responsible for neurovirulence (early paralysis and death) which correlates with elevated levels of infectious virus and the presence of virus antigen within neurons of the brain stem and gray matter of the spinal cord. Both the GDVII and the DA strains of virus contain genetic determinants for late demyelination in spinal cord. However, quantitative analysis of demyelination produced by recombinant GDVII/DA viruses suggest that multiple gene segments influence the number and extent of demyelinating lesions.