The dynamics of Theiler's murine encephalomyelitis virus (TMEV) RNA replication in the central nervous systems of susceptible and resistant strains of mice were examined by quantitative real-time reverse transcription-PCR and were found to correlate with host immune responses. During the acute phase of infection in both susceptible and resistant mice, levels of viral replication were high in the brain and brain stem, while levels of viral genome equivalents were 10-to 100-fold lower in the spinal cord. In the brain, viral RNA replication decreased after a peak at 5 days postinfection (p.i.), in parallel with the appearance of virus-specific antibody responses; however, by 15 days p.i., viral RNA levels began to increase in the spinal cords of susceptible mice. During the transition to and the persistent phase of infection, the numbers of viral genome equivalents in the spinal cord varied substantially for individual mice, but high levels were consistently associated with high levels of proinflammatory Th1 cytokine and chemokine mRNAs. Moreover, a large number of viral genome equivalents and high proinflammatory cytokine mRNA levels in spinal cords were only observed for susceptible SJL/J mice who developed demyelinating disease. These results suggest that TMEV persistence requires active viral replication beginning about day 11 p.i. and that active viral replication with high viral genome loads leads to increased levels of Th1 cytokines that drive disease progression in infected mice.Theiler's murine encephalomyelitis virus (TMEV) is a naturally occurring enteric pathogen of mice that belongs to the Cardiovirus genus in the Picornaviridae family (39, 41). Lowneurovirulence strains, such as BeAn and DA, produce persistent infections in the central nervous systems (CNS) of susceptible strains of mice that result in mononuclear cell inflammation and largely immune system-mediated demyelination, providing an experimental analog for multiple sclerosis (MS) in humans (6,13,19,44). While only small amounts of infectious virus (100 to 1,000 PFU/spinal cord) are recovered during the persistent phase of infection (11, 29), very high levels of viral genome equivalents have been reported (49), suggesting that widespread and persistent TMEV infection involves continuous viral replication with restricted infectious virus production. Viral antigens and RNA are largely found in neurons in the brain and spinal cord during the acute phase of infection (2, 16), whereas macrophages or microglia containing viral antigen(s) and RNA predominate in the spinal cord during chronic infection (3, 30). The spread of infection to oligodendrocytes and astrocytes is also seen (3,9,44,47,51). TMEV replication in macrophages is highly restricted at the levels of RNA replication and virion assembly, consistent with mechanisms of persistence of cytolytic RNA viruses (23, 49). The mechanism(s) underlying TMEV transition from an acute neuronal infection in the gray matter to a chronic macrophagic and glial infection in the white matter is not well u...

The high-neurovirulence Theiler's murine encephalomyelitis virus (TMEV) strain GDVII uses heparan sulfate (HS) as a coreceptor to enter target cells. We report here that GDVII virus adapted to growth in HSdeficient cells exhibited two amino acid substitutions (R3126L and N1051S) in the capsid and no longer used HS as a coreceptor. Infectious-virus yields in CHO cells were 25-fold higher for the adapted virus than for the parental GDVII virus, and the neurovirulence of the adapted virus in intracerebrally inoculated mice was substantially attenuated. The adapted virus showed altered cell tropism in the central nervous systems of mice, shifting from cerebral and brainstem neurons to spinal cord anterior horn cells; thus, severe poliomyelitis, but not acute encephalitis, was observed in infected mice. These data indicate that the use of HS as a coreceptor by GDVII virus facilitates cell entry and plays an important role in cell tropism and neurovirulence in vivo.Theiler's murine encephalomyelitis viruses (TMEV) are divided into two groups based on neurovirulence following intracerebral (i.c.) inoculation of mice. High-neurovirulence strains, such as GDVII, produce fatal encephalitis in mice, while low-neurovirulence strains, such as BeAn and DA, induce early poliomyelitis followed by persistent infection in the central nervous system (CNS) and demyelinating disease in mice (26,28). Members of both neurovirulence groups are 90% identical in nucleotide sequences, 95% identical in amino acid contents, and differ only slightly in their overall threedimensional structures (31). Despite their close genetic and structural relatedness, these viruses differ significantly in their mode of binding to mammalian cells (35,39) and in their association with intracellular membranes during replication (8).TMEV use at least two molecules for attachment and cell entry (19). Recent studies have shown that low-neurovirulence strains bind ␣2,3 sialic acid moieties on N-linked oligosaccharides (7,39,42), while high-neurovirulence strains attach to the proteoglycan heparan sulfate (HS) (35). Brain-derived stocks of GDVII virus bind HS (35), indicating that this interaction is not an artifact of cell culture adaptation of the virus. Since TMEV are picornaviruses, nonenveloped viruses which are unable to fuse with the lipid bilayer of cells, internalization is believed to require a specific protein entry receptor. Viruses of both TMEV neurovirulence groups have been shown to bind an as yet unidentified sialylated 34-kDa protein in a ligand (virus) protein overlay assay (21, 27).To gain further insight into cell entry by TMEV, we focused on the interaction between high-neurovirulence GDVII and HS by using GDVII virus adapted to proteoglycan-deficient cells. The adapted virus, designated GD-A745, acquired two mutations, both on surface loops in the capsid. GD-A745 no longer used HS for binding and entry, and its neurovirulence in mice was attenuated compared to parental GDVII. Neuronal tropism in GD-A745-infected adult mice changed from cereb...

Persistent Theiler's virus infection in the central nervous system (CNS) of mice provides a highly relevant animal model for multiple sclerosis. The low-neurovirulence DA strain uses sialic acid as a coreceptor for cell binding before establishing infection. During adaptation of DA virus to growth in sialic acid-deficient cells, three amino acid substitutions (G1100D, T1081I, and T3182A) in the capsid arose, and the virus no longer used sialic acid as a coreceptor. The adapted virus retained acute CNS virulence, but its persistence in the CNS, white matter inflammation, and demyelination were largely abrogated. Infection of murine macrophage but not oligodendrocyte cultures with the adapted virus was also significantly reduced. Substitution of G1100D in an infectious DA virus cDNA clone demonstrated a major role for this mutation in loss of sialic acid binding and CNS persistence. These data indicate a direct role for sialic acid binding in Theiler's murine encephalomyelitis virus persistence and chronic demyelinating disease.Virus infection in individuals genetically predisposed to multiple sclerosis (MS) is believed to trigger autoimmune myelin damage (32, 46). It is not known whether an acute "hit-andrun" infection suffices or whether infection must be chronic. Theiler's murine encephalomyelitis virus (TMEV) infection of mice, in which persistent central nervous system (CNS) infection induces Th1 CD4 T-cell responses to both virus and myelin proteins, provides a relevant experimental animal model for MS (8,15,37). Cytolytic picornaviruses such as TMEV require continuous virus replication and cell-to-cell spread for persistence; however, virus factors influencing persistence of TMEV or of other RNA viruses remain poorly understood (1, 29).Assembly of recombinant TMEV from sequences of highneurovirulence (nonpersisting) and low-neurovirulence (persisting) strains have mapped a persistence determinant(s) to the capsid, implicating a virus-receptor interaction in CNS persistence (7,19,35). While a protein entry receptor for TMEV has not yet been identified, members of the two neurovirulence groups use different carbohydrate coreceptors: for high-neurovirulence strains, the proteoglycan heparan sulfate, and for the low-neurovirulence strains, ␣2,3-linked sialic acid on an N-linked glycoprotein (14,40,44). Resolution of the structure of low-neurovirulence DA virus cocrystallized with the sialic acid mimic sialyllactose (SLL) demonstrated that sialic acid makes contact with four tightly clustered DA virus capsid amino acids: three on VP2 puff B and the fourth in the VP3/VP1 cleavage dipeptide at the VP3 C terminus (50). These four residues are conserved in all TMEV strains (36), suggesting that sialic acid binding is conformation dependent. Together, these data point to a crucial role for sialic acid coreceptor use by the low-neurovirulence TMEV in CNS persistence.Analysis of DA virus adapted to growth in Lec-2 cells, which lack the CMP-sialic acid transporter (11), revealed three acquired mutations in the capsid (G...