ATTENUATION OF THEILERS MURINE ENCEPHALOMYELITIS VIRUS BY MODIFICATIONS OF THE OLIGOPYRIMIDINE AUG TANDEM, A HOST-DEPENDENT TRANSLATIONAL CIS-ELEMENT

A set of Theiler's murine encephalomyelitis virus mutants with enginee red alterations in the conserved oligopyrimidine/AUG tandem (E. V. Pil ipenko, A. P. Gmyl, S. V. Maslova, G. A. Belov, A. N. Sinyakov, M. Hua ng, T.D.K. Brown, and V.I. Agol, J. Mol. Biol. 241:398-414, 1994) were assayed for their growth potential in BHK-21 cells (as reflected in p laque size) and for neurovirulence upon intracerebral inoculation of m ice. Tandem-destroying mutations, which included substitutions in the oligopyrimidine moiety and extended insertions into the oligopyrimidin e/AUG spacer, exerted relatively little effect on the plaque size but ensured a high level of attenuation. The attenuated mutants exhibited remarkable genetic stability upon growth in BHK-21 cells. However, the brains of rare animals that developed symptoms after the inoculation, vith high doses of these mutants invariably contained pseudorevertants with the oligopyrimidine/AUG tandem restored by diverse deletions or an AUG-generating point mutation. The AUG moiety of the tandem in the revertant genomes was represented by either a cryptic codon or initiat or codon. The results demonstrate that the tandem, while dispensable f or the Theiler's murine encephalomyelitis virus growth in BHK-21 cells , is essential for neurovirulence in mice. Thus, the oligopyrimidine/A UG tandem is a host-dependent cis-acting control element that may be e ssential for virus replication under certain conditions. The functiona l activity of the tandem was retained when its oligopyrimidine or AUG moieties were made double stranded, A possible role of the tandem in t he cap-independent internal initiation of translation on the picornavi rus RNA templates is discussed.