No Thumbnail Available

Influence de la phosphorylation de l’ARN polymérase ARN-dépendante sur la réplication du virus de Theiler.

(2021)

Files

Wieme_Mathilde_61091900_2020-2021.pdf
  • UCLouvain restricted access
  • Adobe PDF
  • 2.09 MB

Details

Supervisors
Faculty
Degree label
Abstract
The Picornaviridae family is composed of small positive-sensed single-stranded RNA viruses. In the laboratory, we work with one of these Picornaviruses: Theiler’s murine encephalomyelitis virus. Mass spectrometry data, from previous experiments, showed that Theiler’s virus RNA-dependant RNA polymerase can be phosphorylated at either a specific serine or a threonine. Sequence alignment indicates that these two residues are highly conserved in Picornaviruses and also in other RNA viruses. Preliminary studies suggested that this post-translational modification blocks the polymerase. Indeed, a phosphomimetic mutation (into aspartate or glutamate) inhibits viral replication and selects revertant viruses. In this project, we wanted to confirm if these previous results were due to the phosphomimetism or to the fact that any mutation that specific localization would affect the structure of the polymerase. To test this, we constructed mutant viruses in which either the serine or the threonine were mutated into any kind of amino acid. We then observed the effect of these mutations on viral replication, using the "split nanoluciferase" system. In addition, we quantified the different viruses and analysed the emergence of revertant viruses. From the quantification of the different mutant viruses, we could say that when the threonine residue was mutated into a phosphomimetic residue, the titer is close to zero. Same results were obtained when the threonine was mutated into a tyrosine or a tryptophan. On the opposite, we found that the asparagine, cysteine and alanine residues seem to be tolerated in that position and don’t seem to have an influence on viral replication. In conclusion, we tried to set up a nanoluciferase system which will allow us to follow the viral replication of the different mutants before the appearance of revertants. Moreover, it seems that phosphomimetic mutations of the threonine and serine residues have an impact on virus replication like tyrosine and tryptophan mutations. While the mutation into a phosphoinhibitor doesn’t seem to influence viral replication. The same can be seen for mutations into tyrosine and tryptophan unlike the alanine mutation (phosphoinhibitor) that doesn’t seem to influence viral replication.