Theiler's Murine Encephalomyelitis Virus L* Amino Acid Position 93 Is Important for Virus Persistence and Virus-Induced Demyelination

The DA strain and other members of the TO subgroup of Theiler's murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection associated with an inflammatory white matter demyelinating disease. TO subgroup strains synthesize an 18-kDa protein, L*, out of frame with the polyprotein from an initiation codon 13 nucleotides downstream from the polyprotein's AUG codon. We previously generated a mutant virus from our infectious DA full-length clone that has a change of the L* AUG codon to ACG (with no change in the polyprotein's amino acid sequence). Studies of this mutant virus showed that L* was key to the TO subgroup phenotype because the mutant had a decreased ability to persist and demyelinate. This work was initially called into question because a similar mutant derived from a different full-length DA infectious clone persisted and demyelinated similarly to wild-type DA virus (O. van Eyll and T. Michiels, J. Virol. 74: 9071-9077, 2000). We now report that (i) the sequence of the L* coding region differs in the two infectious clones, resulting in a Ser or Leu as the predicted amino acid at position 93 of L* (with no change in the polyprotein's amino acid sequence), (ii) the difference in this amino acid is key to the phenotypic differences between the two mutants, and (iii) the change in amino acid 93 may affect L* phosphorylation. It is of interest that this amino acid only appears critical in determining the virus phenotype when L* is present in a significantly reduced amount (i. e., following translation from an ACG initiating codon).