Research Focus

Mechanisms of Theiler's murine encephalomyelitis virus persistence

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Introduction

Theiler's murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus of the family Picornaviridae, is a natural pathogen of mice and is spread horizontally among mice via the gut. Two TMEV groups are based on neurovirulence in mice. The first consists of high-neurovirulence strains that cause a rapidly fatal encephalitis. The other group consists of low-neurovirulence strains that produce a persistent infection of the central nervous system (CNS), leading to an inflammatory demyelinating pathology. Infection with low-neurovirulence TMEV provides a highly relevant experimental animal model for multiple sclerosis (MS), a chronic human neurological disorder in which a viral infection is believed to play a causative role. Investigation of TMEV is facilitated by a small viral genome that is an 8-kb positive-sense, single-stranded RNA molecule, encoding only 12 final gene products. The atomic structures of the virus resolved to ~3Å. Mutations and deletions of the RNA genome can then be performed on cDNAs in vitro and assembled into full-length viral cDNA copies that are infectious for mammalian cells and mice.

Role of TMEV-induced apoptosis in viral persistence

Cell-to-cell spread of TMEV, a lytic RNA virus, is required for persistence in vitro and in vivo. This is in contrast to the persistence of noncytolytic RNA viruses in which the host cell survives. Infection of the principal cell (viral reservoir) in which TMEV persists cannot be highly productive, or viral spread, cell death, and demise of the host will occur. Thus, either (1) selection of genetic viral variants (quasispecies) that are restricted in some replicative function, or (2) the presence of factors in the cell reservoir that restrict wild-type (wt) viral replication is required. TMEV persistence does not result in attenuating mutations. Rather, CNS macrophages in which the TMEV persists restrict viral replication. Although infected macrophages produce on the order of 100 plaque forming units (pfu) of virus per cell at 12 hours post-infection, an 80% reduction in pfu is observed at 16 hr pi as the result of apoptosis. Viral replication causes TMEV-infected macrophages to undergo apoptosis through an intrinsic pathway in differentiated macrophages, causing caspase-3 activation, or the extrinsic pathway in interferon-activated macrophages that involves signaling through receptors for tumor necrosis factor (TNF)- and TNF-related apoptosis-inducing ligand (TRAIL). Numerous apoptotic cells, including macrophages, are found in the inflammatory demyelinating lesions in infected mice. To purse the role of TMEV-induced apoptosis in viral persistence we are: (1) characterizing the intrinsic signaling pathway(s) in cells that restrict TMEV infection and undergo apoptosis, (2) identifying the viral protein(s) that trigger apoptosis, and (3) elucidating the step(s) in the viral life cycle that are restricted in apoptotic cells.

Role of virus receptors in TMEV pathogenesis and persistence

The binding of a virus to the surface of susceptible cells is a major determinant of viral host range and tissue cell tropism. Virus-receptor interactions usually involve multiple steps to promote viral entry and infection. The initial step is often binding to an attachment factor (co-receptor), a low-affinity interaction effecting docking of the virus to the host cell as well as concentrating the virus on the cell surface. Members of the two TMEV neurovirulence groups use different carbohydrate co-receptors: for high-neurovirulence strains the proteoglycan, heparan sulfate and for the low-neurovirulence strains 2,3-linked sialic acid on an N-linked glycoprotein. The structure of low neurovirulence DA virus co-crystallized with a sialic acid mimic revealed that four DA virus capsid residues (three on VP2 puff B and the fourth in the VP2 C-terminus at the VP3/VP1 cleavage dipeptide) 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. Mutation of two of the three residues on VP2 puff B confirmed a role in virus binding. Further, adaptation of DA virus to growth in sialic acid-deficient cells resulted in a virus that no longer used sialic acid as a co-receptor. Although the adapted virus retained acute CNS virulence, persistence in the CNS of mice and inflammation and demyelination were largely abrogated. There are several possible mechanisms to explain TMEV persistence after sialic acid-dependent infection. This is presently under investigation. Since picornaviruses do not have an envelope to fuse with the lipid cell membrane bilayer, binding to a protein entry receptor is necessary for entry and initiation of infection. Thus, a major focus of the lab is the identification of the TMEV protein entry receptor, using biochemical, immunological and molecular biological approaches.