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2019-06-24 - Travail avec promoteur/TFE - Anglais - 55 page(s)

Krynska Hanna, Tafforeau Lionel , "Study of the potential deubiquitination role of PAN2 on PB2, a subunit of Influenza virus polymerase", 2019-06-24

  • Codes CREF : Biologie (DI3100)
  • Unités de recherche UMONS : Biologie cellulaire (S815)
  • Instituts UMONS : Institut des Sciences et Technologies de la Santé (Santé), Institut des Biosciences (Biosciences)

Abstract(s) :

(Anglais) Study of the potential deubiquitination role of PAN2 on PB2, a subunit of Influenza virus polymerase Hanna Krynska, UMONS, June 2019 Flu is a seasonal disease affecting thousands of people each year, which is caused influenza virus. This single-stranded RNA (-) virus has a segmented genome, each segment of which is protected by nucleoproteins and the viral polymerase. It consists of 3 subunits: PA, PB1 and PB2. Recently, the latter has been identified as an interactor of the human protein PAN2. PAN2 is a component of the PAN complex involved in deadenylation of cell mRNAs, function that is provided by its RNAse domain. PAN2 possess another domain: a ubiquitin carboxyl-terminal hydrolase domain (UCH), which is found in deubiquitinases, enzymes removing ubiquitins from their substrates. The PAN2’s UCH domain was considered as non-functional due to a mutation present inside the triad of amino acids essentials for catalytic activity. However, it has recently been shown that PAN2 is a bona fide deubiquitinase. This functional role has been described from a conformational adaptation of PAN2, which, despite the mutation, confers a hydrolase domain. This new role has been the starting point of this master project, which consists of the characterisation of PB2-PAN2 interaction. Several previous experiments have shown that PB2 is ubiquitinated in infected cells and the PAN2 knockdown decreases the influenza polymerase activity. Taking these into account, the aim of research was to focus on the importance of ubiquitins in this interaction, supposing that PAN2 deubiquitinases PB2. The three proteins of interest (PB2, PAN2, Ubiquitin) were overexpressed in human 293T cells. Then, after adaptation of the protein quantification protocol, they were studied by co-immunoprecipitation, followed by a Western blot analysis. We were able to confirm that they are genuine partners, PAN2 being co-precipitated by PB2. However, ubiquitins could not be observed in any of the experiments. As a result, several solutions have been proposed and described in order to deepen the understanding and characterisation of this interaction. Keywords: Influenza virus, viral polymerase, PB2, PAN2, ubiquitin, deubiquitinase