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.     

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.     

Chimeric Theiler's virus with altered tropism for the central nervous system.

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either an acute encephalitis or a persistent demyelinating disease. Following intracranial inoculation, the demyelinating strains infect sequentially the grey matter of the brain, the grey matter of the spinal cord, and finally the white matter of the spinal cord, where they persist and cause chronic demyelination. The neurovirulent strains cause a generally fatal encephalitis with lytic infection of neurons. The study of chimeric Theiler's viruses, obtained by recombining the genomes of demyelinating and neurovirulent strains, has shown that the viral capsid contains determinants for persistence and demyelination. In this article we describe the recombinant virus R5, in which the capsid protein VP1 and a small portion of protein 2A come from the neurovirulent GDVII strain and the rest of the genome comes from the persistent DA strain. The capsid of virus R5 also contains one mutation at amino acid 34 of VP3 (Asn-->His). Virus R5 does not persist in the central nervous system (CNS) of immunocompetent SJL/J or BALB/c mice. However, it replicates efficiently and persists in the CNS of BALB/c nu/nu mice, showing that its growth in the CNS is not impaired. In BALB/c nu/nu mice, whereas virus DA causes mortality with large amounts of viral antigens in the white matter of the spinal cord, virus R5 does not kill the animals, persists in the neurons of the grey matter of the brain, and never reaches the white matter of the spinal cord. This phenotype is due to the chimerism of the capsid and/or to the mutation in VP3. These results indicate that the capsid plays an important role in the characteristic migration of Theiler's virus within the CNS.     

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.     

Assembly of Theiler's virus recombinants used in mapping determinants of neurovirulence.

A major determinant of neurovirulence for the GDVII strain of Theiler's virus, a murine picornavirus, was mapped to the P1 capsid protein region. Chimeric viruses were constructed by using sequences from the 5' noncoding and P1 regions of the virulent GDVII strain to replace equivalent regions of the less virulent BeAn strain. Neurovirulence in mice progressively increased as larger regions of BeAn capsid protein-encoding sequences were replaced. The in vitro growth characteristics of the chimeras showed that some chimeras were growth delayed in BHK-21 cells even though the viral constructs exhibited larger plaque sizes, were less temperature sensitive, and were more thermally stable than BeAn. Examination of assembly intermediates revealed an altered pentamer conformation and delayed empty capsid formation for the growth-compromised viruses. For these constructs, their chimeric nature inadvertently resulted in virion assembly defects that complicated finer-scale mapping of the determinants of virulence within the capsid region. These results demonstrate the importance of determining in vitro growth characteristics of chimeras to correctly decipher the significance of their phenotypes. VP1 does not contain a complete determinate for virulence because a chimera with VP1-encoding sequences from GDVII in an otherwise BeAn virus has an attenuated phenotype but is not growth compromised in vitro. The source of sequences, BeAn or GDVII, in the 5' noncoding region had only slight effects on the virulence of recombinant constructs.     

Role of VP2 amino acid 141 in tropism of Theiler's virus within the central nervous system.

Following intracranial inoculation, Theiler's virus causes either an acute encephalitis (strain GDVII) or a chronic demyelinating disease (strain DA). The DA strain sequentially infects the grey matter of the brain, the grey matter of the spinal cord, and, finally, the white matter of the spinal cord, where it persists in glial cells and causes demyelinating lesions. Analysis of the phenotype of recombinant viruses has shown that the viral capsid contains determinants for persistence and demyelination. Our previous studies showed that a Lys at position 141 of the VP2 capsid protein (VP2-141) could render a chimeric virus persistent. We also reported that another recombinant virus, virus R5, migrated from the grey matter of the brain to that of the spinal cord inefficiently and was unable to infect the white matter of the spinal cord. In this article, we report that introducing a Lys at position VP2-141 in virus R5 increases its ability to infect the white matter of the spinal cord. Our results indicate that this amino acid is important for the spread of the virus within the central nervous system.     

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.     

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.     

Infection of macrophage primary cultures by persistent and nonpersistent strains of Theiler's virus: role of capsid and noncapsid viral determinants

We compared the infection of bone marrow macrophages by the DA and GDVII strains of Theiler's virus and by two viruses constructed by exchanging the DA and GDVII capsids. The replication of the GDVII strain and of both chimeric viruses was restricted in macrophages. Therefore, the infection of macrophages requires both capsid and noncapsid viral determinants.     

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.     

Early infection of the central nervous system by the GDVII and DA strains of Theiler's virus.

The DA strain of Theiler's virus, a murine picornavirus, causes a persistent infection of glial cells of the white matter of the spinal cord, associated with chronic inflammation and primary demyelination. The GDVII strain causes an acute fatal grey matter encephalomyelitis. We characterized the target cells of GDVII and DA viruses 4 days following intracerebral inoculation, and we compared the levels of viral RNA within these cells. GDVII virus infected approximately 10 times more cells than DA virus. Whereas GDVII virus infected neurons exclusively, DA virus infected also astrocytes and possible macrophage-microglial cells. The levels of viral RNA in neurons infected with GDVII and DA viruses were of the same order. These results show that DA virus infects glial cells already at the beginning of the disease and that the more efficient spread of GDVII virus is probably not due to a higher level of RNA replication per cell.     

Localization of a neutralization site of Theiler''s murine encephalomyelitis viruses.

Theiler's murine encephalomyelitis viruses (TMEV) are picornaviruses that produce enteric and neurological diseases in mice. Subgroup TO strains of TMEV cause persistent infections with demyelination, while subgroup GDVII strains neither persist nor demyelinate. We produced neutralizing monoclonal antibodies (mAbs) to clarify the mechanisms of persistence and demyelination. Some of the neutralizing mAbs reacted with isolated VP1 on Western blots, while others were conformation specific. The neutralization site for the former TMEV mAbs was on the VP1 trypsin cleavage site of the intact virion. The neutralization site for the conformation-specific mAbs was distinct and was not affected by trypsin. Trypsin treatment of subgroup TO strains increased their infectivity for L cells, whereas the infectivity of subgroup GDVII strains was decreased by trypsin treatment. Subpopulations of virus in subgroup TO-infected tissue culture cells and in infected mouse brain homogenates contained VP1-cleaved virus; this VP1-cleaved virus gave rise to a large persistent fraction in neutralization tests when it was reacted with VP1-specific mAbs. These findings have implications regarding the pathogenesis of subgroup TO demyelinating disease. TMEV VP1 cleavage may be important for virus persistence because of disruption of a major neutralization epitope. The change in virus surface structure caused by VP1 cleavage may affect cell binding and lead to altered cytotropism. Immunocytes, which have been implicated in subgroup TO demyelination, may provide a source for proteases for VP1 cleavage.     

Prolonged Gray Matter Disease without Demyelination Caused by Theiler's Murine Encephalomyelitis Virus with a Mutation in VP2 Puff B

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups based on neurovirulence. During the acute phase, DA virus infects cells in the gray matter of the central nervous system (CNS). Throughout the chronic phase, DA virus infects glial cells in the white matter, causing demyelinating disease. Although GDVII virus also infects neurons in the gray matter, infected mice developed a severe polioencephalomyelitis, and no virus is detected in the white matter or other areas in the CNS in rare survivors. Several sequence differences between the two viruses are located in VP2 puff B and VP1 loop II, which are located near each other, close to the proposed receptor binding site. We constructed a DA virus mutant, DApBL2M, which has the VP1 loop II of GDVII virus and a mutation at position 171 in VP2 puff B. While DApBL2M virus replicated less efficiently than DA virus during the acute phase, DApBL2M-induced acute polioencephalitis was comparable to that in DA virus infection. Interestingly, during the chronic phase, DApBL2M caused prolonged gray matter disease in the brain without white matter involvement in the spinal cord. This is opposite what is observed during wild-type DA virus infection. Our study is the first to demonstrate that conformational differences via interaction of VP2 puff B and VP1 loop II between GDVII and DA viruses can play an important role in making the transition of infection from the gray matter in the brain to the spinal cord white matter during TMEV infection.     

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.     

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.     

Molecular Characterization of a Nondemyelinating Variant of Daniel’s Strain of Theiler’s Virus Isolated from a Persistently Infected Glioma Cell Line

Wild-type Daniel’s strain of Theiler’s virus (wt-DA) induces a chronic demyelination in susceptible mice which is similar to multiple sclerosis. A variant of wt-DA (designated DA-P12) generated during the 12th passage of persistent infection of a G26-20 glioma cell line failed to persist and induce demyelination in SJL/J mice. To identify the determinants responsible for this change in phenotype, we sequenced the capsid coding sequence (nucleotides [nt] 2991 to 3994) and found three mutations in VP1: residues 99 (Gly to Ser), 100 (Gly to Asp), and 103 (Asn to Lys). To study the role of these mutations in neurovirulence and demyelination, we prepared a recombinant virus, DAP-1C-2A/DA, with replacement of wt-DA nt 2991 to 3994 with the corresponding region of DA-P12, and viruses with individual point mutations at VP1 residues 99(Ser), 100(Asp), and 103(Lys). DAP-1C-2A/DA and viruses with a mutation at VP1 residue 99 or 100 (but not 103) completely attenuated the ability of wt-DA to induce demyelination. Failure to induce demyelination was not due to a general failure in growth, since DA-P12 and other mutant viruses lysed L-2 cells in vitro as effectively as wt-DA. The change in disease phenotype was independent of the specific B- or T-cell immune recognition because a decrease in the neurovirulence of mutant viruses was observed in neonatal mice and immune-deficient RAG1 −/− mice. This difference in neurovirulence is not the complete explanation for the failure of DA-P12 to demyelinate, since virus with a mutation at residue 103(Lys) had decreased neurovirulence but did induce demyelination. Therefore, point mutation at VP1 residue 99 or 100 altered the ability of wt-DA to demyelinate, perhaps related to a disruption in interaction between virus and receptor on certain neural cells.     

Theiler's virus and Mengo virus induce cross-reactive cytotoxic T lymphocytes restricted to the same immunodominant VP2 epitope in C57BL/6 mice.

C57BL/6 mice develop a virus-specific cytotoxic T-lymphocyte (CTL) response after intraperitoneal inoculation with either the DA strain of Theiler's virus or Mengo virus, two members of the Cardiovirus genus. These CTLs contribute to viral clearance in the case of Theiler's virus but do not protect the mice from the fatal encephalomyelitis caused by Mengo virus. In this study we show that DA and Mengo virus-induced CTLs are cross-reactive. The cross-reactivity is due to a conserved, H-2Db-restricted epitope located between amino acid residues 122 and 130 of the VP2 capsid protein (VP2(122-130)). This epitope is immunodominant in C57BL/6 mice infected with Theiler's virus. The VP2(122-130) epitope, initially identified for Mengo virus, is the first CTL epitope described for Theiler's virus.     

Replacement of Loop II of VP1 of the DA Strain with Loop II of the GDVII Strain of Theiler’s Murine Encephalomyelitis Virus Alters Neurovirulence, Viral Persistence, and Demyelination

Theiler’s murine encephalomyelitis viruses, which are murine picornaviruses, can cause central nervous system inflammatory disease. To study the role of loop II in capsid protein VP1, two mutant viruses of strain DA in which DA loop II amino acids were replaced with strain GDVII amino acids were constructed. Infection of mice with the two mutant viruses led to dramatically different patterns of disease.     

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.