Virus Persistence in an Animal Model of Multiple Sclerosis Requires Virion Attachment to Sialic Acid Coreceptors

Abstract

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...

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

“…3C), but viral copy numbers were dramatically reduced (>10 6 -fold) (Fig. 3D) and white matter inflammation and demyelination in the spinal cord were significantly reduced at 55 days pi [25]. A role for VP1 loop II residue, G1100D, in sialic acid binding and CNS persistence was demonstrated by analysis of a DA infectious cDNA clone with this mutation (Fig.…”

“…Since the capsid conformation of the VP2 puff B in the low-neurovirulence strains appears to be responsible for sialic acid binding, we asked whether a low-neurovirulence TMEV that did not use sialic acid as a co-receptor would retain the ability to cause persistent infections. To generate a DA virus that did not use binding to sialic acid for infection, the parent virus was passed in Lec-2 cells, which lack the CMP sialic acid transporter and cell surface sialic acid [25]. DA virus was used because of the availability of the original brain-derived virus stock that had never been passed in cell culture.…”

Mouse as a Model Organism

“…Histopathology Findings: Depending on the type or cause, there may be variation in fiber size, varying degrees of myofiber degeneration, myocytolysis, loss of striation, internal nuclei; +/-inflammation; regeneration with internal nuclei in tandem array or replacement fibrosis with fibrocollagen (Vainzof et al, 2008). Deb et al, 2009;Kumar et al, 2004;Lavi et al, 1986;Tsunoda et al, 1996;Turrin, 2008;Woyciechowska et al, 1984). Nurture Influences: Diet and drug interventions may modulate some myopathy and muscular dystrophy phenotypes (Girgenrath et al, 2009;Zdanowicz et al, 1995).…”

“…Nature Influences (Vainzof et al, 2008): Dysferlin mutations in A/J and SJL/J mice (Ho et al, 2004;Wenzel et al, 2007); spontaneous mutations in Lama2 (merosin or laminin, alpha2) in 129/Re mice (Jasmin and Bajusz, 1962;Zdanowicz et al, 1995), or in Dystrophin in (Mdx) mice (Lefaucheur, Pastoret, & Sebille, 1995). Nature Influences: Susceptibility to infection or disease (Azoulay-Cayla et al, 2001;Coley et al, 2005;Dandekar & Perlman, 2002;Deb et al, 2009;Kumar et al, 2004;Lavi et al, 1986;Tsunoda et al, 1996;Turrin, 2008;Woyciechowska et al, 1984). Deb et al, 2009;Kumar et al, 2004;Lavi et al, 1986;Tsunoda et al, 1996;Turrin, 2008;Woyciechowska et al, 1984).…”

Mutation of the Theiler's virus leader protein zinc-finger domain impairs apoptotic activity in murine macrophages

“…The mutated L sequence in a subclone in pGEM13 was then assembled as previously described (Kumar et al, 2004) into a full-length BeAn virus infectious clone to generate a progeny virus stock. Although the one-step growth kinetics in M1-D macrophages was similar for the two viruses and the mutant virus infection produced slightly higher numbers of pfu/cell at 8 to 12 h pi (Fig.…”