PhD PROPOSAL FOR THE PASTEUR-PARIS UNIVERSITY INTERNATIONAL PROGRAM

 

Deadline for full application: December 15th, 2013

 

Interviews: March, 2014

Start of the Ph.D.: October 1st, 2014

 

 

 

Department: Virology

Title of the PhD project: Control of influenza A virus mRNA splicing by cellular splicing factors: a potential target for antiviral therapeutics

Name of the lab: Molecular Genetics of RNA Viruses

Head of the lab: Sylvie van der WERF

PhD advisor: Nadia NAFFAKH

Email address: nadia.naffakh@pasteur.fr

Web site address of the lab: http://www.pasteur.fr/fr/recherche/virology/units-groups/molecular-genet...

Doctoral school affiliation and University: B3MI – University Paris Diderot-Paris 7

 

Presentation of the laboratory and its research topics:

Our laboratory associates three research groups on influenza viruses and one research group on hepaciviruses. It is also a National Influenza Center for Northern France and a WHO H5 reference laboratory. Our activities include studies on viral evolution, mechanisms of expression and replication of the viral genomes, and analysis of virus-host interactions.

To identify influenza virus-host protein-protein interactions that determine virulence and host-range, we are using methods based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection [1,2], as well as high-throughput technologies including state-of-the-art yeast two-hybrid and a split-luciferase based complementation assay in human cells [3]. Functional studies are performed on selected subsets of viral-host protein-protein interactions. We are currently focusing on the interplay of the viral polymerase heterotrimer PB1-PB2-PA with three areas of the human proteome : the RNA processing, nucleo-cytoplasmic trafficking, and ubiquitin proteasome pathways.

Description of the project:

 

Background: Unlike most viruses with an RNA genome, influenza A viruses replicate in the nucleus of infected cells, and their replication is highly reliant on host cell transcriptional and splicing machineries. The M and NS genomic segments produce unspliced (M1, NS1) and spliced (mainly M2 and NS2) mRNAs. We recently identified a component of the human spliceosome as an interacting partner of influenza virus polymerase, through a yeast two-hybrid screen. We obtained several lines of evidence that this factor associates with the viral polymerase in infected cells. The specific depletion of this factor in infected cells led to reduced amounts of the spliced transcript encoding NS2, to a reduced NS2/NS1 protein ratio, and to a strongly impaired production of infectious influenza virions. Overall, our results show that this splicing factor, referred to as SF below, promotes influenza virus replication by controlling the splicing of the viral NS1 mRNA and the production of the essential NS2  protein.

 

Project: we aim to understand the mechanism through which SF, and possibly SF-associated splicing factors, control the splicing of influenza A virus mRNAs. We also aim to explore whether SF interacts directly with the viral RNA and/or viral polymerase, assuming that such interactions represent potential targets for the development of therapeutic antiviral agents.

 

a) We will investigate which viral components cooperate with SF to regulate the splicing of NS1 mRNA. To determine whether the viral polymerase is involved, the NS1 mRNA will be expressed transiently, either under the control of the viral polymerase as described earlier [4], or under the control of the cellular RNA polymerase II. The effect of SF depletion will be assessed in both systems, by quantifying the levels of NS2/NS1 mRNAs and proteins by RT-qPCR and western blot. We will further examine whether SF-mediated control of NS1 splicing is dependent i) on the presence of the NS1 protein (by comparing the wild-type mRNA and a ∆NS1 mRNA with a stop codon in NS1 open reading frame) ; and ii) on the strength of the NS2 5’ splice site (by comparing the wild-type mRNA and an NSopt mRNA with an optimized 5’ splice site).

 

b) In parallel, we will determine whether SF binds directly to the viral NS1 mRNA, using biochemical analyses by as formaldehyde and UV cross-linking, and we will characterize further SF’s interaction with the PB2 subunit of the viral polymerase. A split-luciferase based complementation assay will be used to define the domains of SF and PB2 involved. We will select mutants of PB2 which are unable to interact with SF by a reverse two-hybrid system in yeast using a library of random mutants of PB2 recently produced in our lab. Mutations which preserve viral polymerase activity in a transient expression assay will be introduced in a reverse genetics system, and the corresponding recombinant viruses will be produced and characterized. Mutations leading to a defect of NS1 mRNA splicing and to viral attenuation would point to an essential role of PB2-SF interaction in the control of NS1 mRNA splicing, and to critical residue(s) of PB2.

 

c) To broaden our understanding of the control of NS1 mRNA splicing, we will also focus on a subset of spliceosomal proteins which were recently shown to interact directly or closely with SF [5]. The effect of silencing these proteins on NS1 mRNA splicing and on the production of infectious virions will be examined. Proteins of interest will be studied further in the transient NS1 mRNA expression systems described above and tested for their ability to bind the viral NS1 mRNA and polymerase, in order to understand how they cooperate with SF.

 

References:

1. Munier S, Rolland T, Diot C, Jacob Y, Naffakh N (2013) Exploration of binary virus-host interactions using an infectious protein complementation assay. Molecular & Cellular Proteomics, PMID: 23816991.

2. Ge X, Rameix-Welti MA, Gault E, Chase G, Dos Santos Afonso E, Picard D, Schwemmle M, Naffakh N (2011) Influenza A virus infectioni induces the nuclear relocalization of the Hsp90 co-chaperone p23 and inhibits the glucocorticoid receptor response. PLoS One, PMID: 21853119  

3. Cassonnet P, Rolloy C, Neveu G, Vidalain PO, Chantier T, et al. (2011) Benchmarking a luciferase complementation assay for detecting protein complexes. Nature Methods 8: 990-992.

4. Robb NC, Jackson D, Vreede FT, Fodor E (2010) Splicing of influenza A virus NS1 mRNA is independent of the viral NS1 protein. The Journal of General Virology 91: 2331-2340.

5. Hegele A, Kamburov A, Grossmann A, Sourlis C, Wowro S, et al. (2012) Dynamic protein-protein interaction wiring of the human spliceosome. Molecular Cell 45: 567-580.

 

 

Keywords:

Influenza virus - virus host-interaction - splicing factors - viral polymerase - viral RNA processing

 

 

Expected profile of the candidate (optional):

We are looking for a highly motivated student, team-oriented and willing to actively participate in the life of the lab. Good oral and written skills in english and a background in molecular biology will be appreciated.

 

 

Contact

Nadia Naffakh

nadia.naffakh@pasteur.fr

33-1-45 68 88 11

Mis à jour le 16/09/2013