DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Sequence and structure of the packaging signals of the influenza A virus genome – PSiFlu

RNA, the engine of flu virus assembly

Sequence and structure of the packaging signals of the influenza A virus genome. The genome of influenza A viruses consists of 8 vRNAs that associate to a polymerase complex and multiple copies of nucleoprotein. The 8 resulting ribonucleoproteins are selectively incorporated in the viral particles thanks to the presence of packaging signals on each vRNA.

Exhaustive high resolution mapping of the packaging signals and role of the intermolecular vRNA interactions in the selective packaging of the segmented genome of influenza A viruses

Influenza A viruses (IAVs) are responsible for recurrent flu epidemics and occasional devastating pandemics in human and animal populations. In addition to seasonal flu, which claims 250,000 to 500,000 lives yearly, new IAVs with high pandemic potential regularly emerge. Evolution of seasonal viruses and emergence of pandemic viruses are greatly facilitated by the IAV segmented genome consisting of 8 negative sense vRNAs, which allows rapid evolution by genetic reassortment but complicates viral assembly, as IAVs must incorporate at least one copy of each vRNA to be infectious. The first step in the generation and propagation of new reassortant viruses, including some with pandemic potential against which humans have limited or no pre-existing immunity, is the co-packaging of vRNAs from two different parental viruses infecting the same cell into a single progeny viral particle. Thus, it is not possible to understand the genesis of reassortant IAVs without first understanding the molecular details of vRNA packaging in the parental viruses. The aim of our project is to unravel the molecular mechanisms that control packaging of the genome of IAVs. The prevailing hypothesis is that interactions between packaging signals of different vRNAs govern their selective packaging. We will extensively and precisely map these packaging signals, then we will identify and validate intermolecular vRNA/vRNA interactions and test their role in viral replication, co-packaging of the vRNAs and viral assembly. Finally, we will test the robustness of the vRNA interaction network. In addition to its fundamental importance, our project could have enormous impact on human - and animal - health, by greatly improving the process of vaccine production and helping to predict the emergence of pandemic strains.

The packaging signals are identified exhaustively at single nucleotide resolution by MIME (Mutational Interference Mapping Experiment), an innovative method recently developed by one of the partners (Smyth et al. Nature Methods 2015). MIME is based on i) the introduction of random point substitutions in each of the 8 vRNAs, ii) the expression of the pool of mutated vRNA molecules in cells using a modified reverse genetic system, iii) the purification of the vRNAs from cells and progeny virions, iv) the sequencing of each pool using next generation sequencing, and v) the analysis of the mutation frequency of each nucleotide in each fraction. The underlying idea behind MIME is that mutations that interfere with the incorporation of vRNAs into virions will be under-represented in the virions compared to cells. MIME identifies packaging signals as well as potential base-pairings between vRNAs. These are tested using trans-complementary mutations that destroy or restore base-pairing in vitro and in a reverse genetic system. The importance of these intermolecular interactions can then be tested on the viral and HA titers, as well as the replication kinetics and packaging of the vRNAs. Packaging is analyzed by RT-qPCR, competition experiments and electron microscopy. Viral assembly at the budding site is studied by cryo-CLEM on entire frozen cells. Finally, the robustness of the RNA interaction network is analyzed by electron tomography, which identifies vRNPs within viral particles, combined in vitro analysis of the vRNA-vRNA interactions by electrophoresis under native conditions.

In order to apply MIME to influenza A viruses, we constructed a completely new reverse genetic system: it consists of a minimal number of plasmids and ensures that all cells that are able to replicate the vRNA library will express the complete set of vRNAs and proteins required to produce viral particles. In our hands, this new reverse genetic system produces significantly more viral particles than widely used reverse genetic system. In parallel, the three partners set up and developed the tools and strategies required for the remaining steps of the project. One of these tools is a user-friendly bioinformatic pipeline that allows analysis of MIME data by non-specialists and has been published in Bioinformatics. It is not only useful for the PSiFlu project, but is also available to all members of the scientific community who would like to apply MIME to other biological questions. Mutant libraries of each of the genomic segments containing 0.5 to 1 % of random substitutions have been obtained. They have been introduced in reverse genetic vectors, plasmids or linear amplicons. Production of viral particles, as well as their purification on red blood cells have been optimized. Wild type PR8 viruses have been produced and analyzed by electron tomography, revealing new details of the internal organization of the influenza A virions. Reassortant vaccines viruses have also been produced and the interaction between the “foreign” vRNAs and the PR8 vRNAs have been analyzed in vitro.

Optimization of the production of viral particles containing the mutant vRNA libraries is in its final stage and the whole set of viruses will be purified within the next few months. At this stage, the vRNA will be extracted from the viral particles and from the producer cells, reverse transcribed, and sequenced using next generation sequencing in order to precisely determine the mutation frequency at each position of each vRNA in both populations. Mutations underrepresented in the viral particles will be those that have a negative impact on the incorporation of vRNAs and will allow the identification of the packaging signals. Complementarities between these signals will be used by bioinformatics, and this will all to establish a list of candidate interactions between vRNAs potentially involved in the packaging of these vRNAs. The existence and the function of these interactions will be tested by reverse genetics, via trans-complementary mutations that will destroy and restore them. The impact of these interactions will be tested on viral replication, vRNA packaging, assembly, and internal architecture of the viral particles. Altogether, this data will establish whether interactions between vRNAs play a general and central role in vRNA packaging and viral particle assembly. In the long term, using the same strategy, it will be possible to study the role of the intermolecular vRNA interactions in genetic reassortment. This could have a significant impact on the production of flu vaccines and on the evaluation of the risk of emergence of reassortant viruses with a high pandemic risk.

Original article in peer-review journal:
- Smith, M.R., Smyth, R.P., Marquet, R. & von Kleist, M. (2016). MIMEAnTo – Profiling functional RNA in Mutational Interference Mapping Experiments. Bioinformatics 32, 3369-3370.

Presentations at meetings:
- Contrant, M., Rosa-Calatrava, M. Bron, P., Marquet, R. & Smyth, R.P. Cartographie à haute résolution des signaux d’empaquetage des ARN viraux des virus influenza de type A par MIME (Mutational Interference Mapping Experiment).
XIX Journées francophones de virologie, Paris (France), 30-31 mars 2017.

- Contrant, M., Rosa-Calatrava, M. Bron, P., Marquet, R. & Smyth, R.P. High-resolution mapping for Influenza A RNA packaging signals by MIME (Mutational Interference Mapping Experiment)
The Sixth ESWI Influenza Conference, Riga (Lituanie), 10-13 septembre 2017.

The aim of our project is to unravel the molecular mechanisms that control packaging of the genome of Influenza A viruses (IAVs). In addition to its fundamental importance, it could have enormous impact on human - and animal - health, by greatly improving the process of vaccine production and helping to predict the emergence of pandemic strains. Influenza A viruses are responsible for recurrent flu epidemics and occasional devastating pandemics. Evolution of seasonal viruses and emergence of pandemic viruses are greatly facilitated by the IAVs segmented genome consisting of 8 (-) sense RNAs (vRNAs), which allows rapid evolution by genetic reassortment but complicates its assembly, as IAVs must incorporate at least one copy of each vRNA to be infectious. The prevailing consensus is that vRNAs are selectively packaged into budding virions. This model has important implications for genetic reassortment and for the design and production of vaccines, yet little is known about the molecular mechanisms underlying specific packaging. Whilst the existence of cis-acting packaging signals has been demonstrated in all 8 vRNAs, they have only been incompletely delineated. A longstanding hypothesis is that direct vRNA/vRNA interactions within packaging signals govern selective packaging but progress in demonstrating it has been slow and limited.
In an on-going collaboration to address this issue, the Marquet and Rosa-Calatrava teams combined in vitro assays with virology approaches and electron tomography to demonstrate that the 8 vRNAs of IAVs build up a single network of interactions. Importantly, they further showed that an in vitro identified vRNA/vRNA interaction is required for optimal replication of the H5N2 IAV and is involved in the selective co-packaging of the interacting vRNAs. This is the strongest evidence so far indicating that the “vRNA/vRNA interaction hypothesis” is correct.
We will extensively and precisely define the vRNA primary and secondary structures of all the cis-acting IAV packaging signals. Rather than testing the effect of a limited number of mutations, usually large deletions, on viral replication, we will use a multidisciplinary approach combining molecular biology (Marquet and Rosa-Calatrava teams), virology (Marquet and Rosa-Calatrava teams) and microscopy (Rosa-Calatrava and Bron teams). First, we will apply an innovative systems biology approach (Marquet team, Smyth et al., revised manuscript under review at Nature Methods), based on the random mutation of each nucleotide of each vRNA and selection of the functional molecules by the viral machinery itself, to define, in an exhaustive and unbiased manner, the primary and secondary structure elements of each vRNA important for packaging. We will then identify and validate intermolecular vRNA/vRNA interactions and test their functional role in viral replication, co-packaging of the partner segments and viral assembly, using in vitro, in cellulo and in viro approaches. The influenza A/PuertoRico/8/34 (H1N1) strain (PR8), used for vaccine production, will be our working model. Finally, the robustness of the vRNA interaction network will be tested by electron tomography, by comparing the internal organization of PR8 virions with that of reassortant viruses where PR8 segments have been replaced by foreign segments of vaccinal interest.
Our ultimate goal is to understand the rules underlying genetic reassortment of IAVs. This research will help to evaluate the likelihood of generating reassortant viruses, including those with pandemic potential. Importantly, successful completion of this project should also pave the way for improving the generation, selection and replication of reassortant viruses used for vaccine production. The strength of the proposal relies on the existing collaboration between the Marquet and Rosa-Calatrava teams and on the Bron team, with specific expertise in structural analysis of viruses.

Project coordination

Roland Marquet (Architecture et Réactivité de l'ARN)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSERM U1054 / CNRS UMR5048 Centre de Biochimie Structurale
UPR 9002 CNRS Architecture et Réactivité de l'ARN
VIRPATH Laboratoire de Virologie et Pathologie Humaine VirPath

Help of the ANR 502,000 euros
Beginning and duration of the scientific project: October 2015 - 36 Months

Useful links

Explorez notre base de projets financés

 

 

ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter