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Modulation of RNA-based regulatory processes by viruses

Periodic Reporting for period 3 - RegulRNA (Modulation of RNA-based regulatory processes by viruses)

Reporting period: 2019-01-01 to 2020-06-30

Our understanding of the importance of non-coding (nc) RNAs in various biological processes has grown exponentially in the past few years. Because they are so preponderant, it is no wonder that virtually every biological function is at one point or another regulated by these molecules. Although they vary a lot in their origin, biogenesis and mode of action, we can broadly distinguish between two types of ncRNAs based on their size. At one end of the spectrum are small ncRNAs, which are mostly in the range of 20-30 nucleotides (nt), on the other side, we can find long ncRNAs, which are every other RNAs with a size arbitrarily defined above 200 nt. Ever since research on ncRNAs has started, their importance in the context of viral infection has been the subject of intense scrutiny. Indeed, the very nature of viruses makes them strictly dependent on a host cell to be able to replicate, translate their mRNAs, and ultimately generate new viral particles to spread to the next host. In the frame of this project, we study how a viral infection can impact pathways involving small ncRNAs. The viruses we study are associated with a number of diseases and constitute a threat to human health. There are two types of viruses that are the focus of our research: i) DNA viruses, such as the oncogenic Kaposi’s sarcoma herpesvirus (KSHV) or the mouse cytomegalovirus (MCMV), and ii) arthropod-borne RNA virus, such as Sindbis virus or Chikungunya virus. The results of our research will help us to better understand the molecular mechanisms at play during the viral replication cycle and how the infected organism responds to the infection at the cellular level. Eventually, we might be able to design new antiviral therapeutic approaches to fight some of these pathogens.
The objectives of our proposal are multifold and pertain either to the understanding of the regulation of one family of small RNAs, coined microRNAs (miRNAs), during viral infection, or to decipher the importance of a mechanism of antiviral defense relying on the generation of small interfering RNAs (siRNAs) by the cell. The three main work packages of the project are:
1. To understand how the biogenesis of virus-encoded miRNAs can be regulated by specific cofactors that can bind to the RNA molecule to modulate its processing by ribonucleases Drosha and Dicer
2. To decipher the molecular machinery at play in the degradation of mature miRNAs, and to identify their roles in viral infections
3. To resolve the long-standing question of the antiviral role of RNA silencing in mammals by identifying regulators of this activity
We have made good progress in WP1 and 3, and we are now close to getting publishable results for these. We have experienced some delays in WP2 but have recently obtained promising and unexpected results that we are working hard to confirm. Here are the details of the main results obtained per work package.
-WP1. Regulation of miRNA biogenesis
The biogenesis of miRNAs requires the sequential action of two type III ribonucleases, Drosha and Dicer, on a long primary transcript (the pri-miRNA) to give rise to an RNA duplex. One of the two strands of this duplex is then assembled into a protein from the Argonaute family to form the functional mature miRNA that can act on target RNAs. This process is highly regulated at every step, and some proteins have been shown to act by binding directly to the pri-miRNA, or by interacting with Drosha or Dicer to modulate their efficiency. In this project, we have set to study the regulation of miRNA biogenesis using as a model a pri-miRNA transcript of viral origin. Namely, we are working with KSHV, which genome contains a unique cluster of twelve miRNAs grouped on a single pri-miRNA. Although all KSHV miRNAs derive from the same transcript, the relative abundance of each individual mature viral miRNA varies a lot, which indicates that some post-transcriptional regulation takes place. The first part of this WP was dedicated to the analysis of the importance of the secondary structure of KSHV primary miRNA transcript in its cleavage efficiency by Drosha. As of now, we have not yet started this analysis, but we are generating a cell line knocked-out of Drosha, which is an essential pre-requisite to go on with this part.
We have however made very good progress regarding the second part of this work package, which consists in a proteomics analysis of putative proteins binding individual KSHV miRNA stem loop precursors (pre-miRNA). We have designed a novel approach to pull-down proteins (see Research and technological achievements) binding to synthetic pre-miRNA of either viral or cellular origin. We now have data from more than eighty mass spectrometry runs that we are in the process of validating. We found at least one specific proteins for each KSHV miRNAs that should allow us to explain how the biogenesis of viral miRNAs is regulated. As soon as we have validated the role of these proteins, we will submit a manuscript for publication.

-WP2. Interplay between miRNA and long ncRNA and mechanism of miRNA decay.
One pre-requisite for this WP was to identify novel examples of cellular miRNA regulation upon virus infection. We decided to look at this using a slightly different approach, i.e. identifying first cellular miRNAs that can regulate virus infection. Indeed, we previously found one cellular miRNA, miR-27, that could down-regulate the mouse cytomegalovirus (MCMV), and later showed that the virus counteracts by inducing degradation of this miRNA. In order to get a broader picture of the involvement of cellular miRNAs during viral infection, we have performed a genome wide screen to look at the effect of over-expressing or inhibiting each human miRNA individually on viral infection. We made use of an engineered Sindbis alphavirus (SINV) expressing GFP to easily monitor the impact on virus level. Although we did not find new evidence of cellular miRNAs negatively regulating SINV, we found a handful of miRNAs that positively regulate this virus. Very interestingly, among these was the neuron-specific miR-124. Given that SINV can sometimes be linked to encephalitis, this is a very promising finding. We are currently working toward the elucidation of the molecular mechanism through which miR-124 can positively regulate SINV, and we also got preliminary evidence that it can also act on Chikungunya virus. We plan to submit these results for publication before the end of the year.
In addition to this, we have continued our work on the functional characterization of the mechanism behind target RNA dependent miRNA decay (TDMD) observed during MCMV infection. Since we had previously identified key players in this pathway, we are now focusing on their complete characterization. Initially, we wanted to study enzymes involved in the modification of miRNA by addition of nucleotides at their 3’ extremity, but given the high redundancy of these in the human genomes (there are seven different terminal nucleotidyl transferases), we focused our efforts on the ribonuclease that can degrade small RNAs. We have recently obtained cell lines expressing an endogenously tagged version of the DIS3L2 protein, either wild type or catalytically inactivated, that will prove extremely valuable to fully characterize this protein.
Finally for this WP, we took an alternative approach to identify other players involved in TDMD, but also in the assembly of miRNAs into Argonaute proteins. Basically, we are using an in vivo biotinylation approach (see later in Research and technological achievements) to find partners of the mouse Argonaute 2 protein during infection by MCMV.

-WP3. Regulation of innate immunity and antiviral RNAi
For this part of the project, we initially wanted to study the role of an heterologous protein, the Drosophila Dicer-2 protein, in antiviral response when expressed in mammalian cells. The rationale behind this idea came from previous results obtained in the laboratory, which showed that the expression of Dicer-2 rendered the cells more susceptible to viral infection rather than providing a protection mediated by degradation of the viral RNAs. We also reported that this proviral effect appeared to be the consequence of a competition between Dicer-2 and double-stranded (ds) RNA sensors such as the protein kinase R (PKR) that are normally activated upon sensing viral RNA replication intermediates. More generally, the idea was also to understand whether antiviral RNAi could play a physiological role in mammals or if it was completely replaced by the interferon response. At the beginning of the project, we decided to drop the study of Dicer-2 in mammalian cells because it was quite artificial. Rather, we have now generated stable cell lines that are knocked-out of the endogenous Dicer and expressed a tagged version. We used this useful tool to immunoprecipitated the tagged Dicer protein in non-infected and virus-infected cells, in order to identify specific co-factors interacting with Dicer specifically in response to virus infection. We have now obtained very interesting results that are detailed later, and which show that known dsRNA sensors interact with Dicer only in infected cells. This indicates that Dicer could play a role in sensing viral RNA, but rather than actively degrade it, can help to recruit other antiviral factors. This part of the project is about to be written up and will be submitted for publication soon.
In parallel to this Dicer-centered approach, we are also tackling this question from a completely unbiased point of view. Since we would like to know whether there are some factors that can restrict the action of RNAi in mammalian somatic cells, we have decided to globally identify proteins involved in the sensing of dsRNA and in the cellular response to this molecule. To this end, we have developed two unrelated but complementary approaches. The first one consists in pulling-down dsRNA using an antibody that specifically recognizes dsRNA molecules and to then analyze proteins that are bound to dsRNA using mass spectrometry. The other approach is more a functional one and is based on a genome-wide CRISPR-Cas9 survival screen aiming at identifying genes involved in the cell death induced by dsRNA transfection. Both approaches have now yielded the first results and we are in the process of validating them.
As detailed above, we have made significant progress in all the different parts of this project. The novel findings that go beyond the state of the art are:
-The comprehensive identification of proteins binding specifically to virus-encoded miRNA precursors to regulate their processing. This is especially relevant since one of KSHV miRNA is a well-known orthologue of the oncogenic cellular miRNA miR-155, and it is of prime interest to understand the specificity of expression of these two related miRNAs. In addition, it should also help us to understand whether viral miRNAs really are completely undistinguishable from cellular ones when it comes to their maturation by the cell machinery. By the end of the project, we will have published a complete list of proteins regulating KSHV miRNA biogenesis and will have fully characterized the mode of action of some of them. We will also be able to understand the importance of pri-miRNA secondary and tertiary structure for its recognition by these RNA binding proteins.
-The identification of the neuron-specific miR-124 as a proviral miRNA during alphavirus infection. Until now, there was only very limited evidence that cellular miRNAs could play positive roles for viruses, and our findings indicate that the tissue specificity of some miRNAs could also dictate the tropism of specific viruses. By the end of the project, we will have nailed the molecular mechanism underlying this effect and initiated the exploitation of this finding to design antiviral therapeutics.
-Finally, the determination of interactors of the Dicer protein during viral infection indicates that there are still new functions of this protein to be discovered. We are very near to complete the characterization of these interactions and to decipher their functional implication during infection. We have also started to extend these observations to other viruses, such as Dengue or Zika virus. The unbiased approaches that we have launched are also bringing new answers regarding the sensing of dsRNA generated during viral infection, and until the end of the project we will dramatically have improved our understanding of the mechanisms at play in the initial steps of a virus infection.