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You are here: Home / Teams / Ohlmann T - REVE / Team projects

Team projects

Infection of the host-cell by viruses induces multiple changes that perturb gene expression. We study these modifications at the level of post-trancriptionnal gene expression both by looking at viral and cellular RNAs.

Research Activities

 

As depicted below, our research focuses on translational control (WP1 and WP2), the development of innovative tools for RNA based studies (WP3), m6A modifications of the mature RNA and the role of alternative splicing in the context of EBV infected cells (WP4).

 

 

WP 1-Structure function of IRES (Internal Ribosomes Entry Segments)

(a) The HIV-1 IRES

Translation of the HIV-1 unspliced genomic mRNA can take place by the canonical cap-dependent mechanism from the 5'-end of the mRNA with the assistance of DDX3 or it can be driven by an  IRES element located in beginning of the Gag coding region. This IRES was initially identified as it drives the production of a shorter Gag isoform of 40 kDa that we can detect very well by in vitro translation but which is more difficult to evidence in HIV-1 infected cells. We are currently investigating the molecular mechanisms of p40 production and its role in viral replication.

(b) Assessing the role of the IRES mechanism in vivo

Using the Nanoblades (CRISPR/Cas9 VLP delivery system), we are investigating the mechanism of IRES-driven protein synthesis into cells. For this, we have several approaches that are developped in parallel: (i) Systemic mutations/deletions of cellular IRESes and their effect on protein synthesis; (ii) design and engineering of an inducible artificial IRES.

 

WP 2-The role of trace elements in RNA metabolism, translation and viral infection

 (a) Selenium, selenoproteins and HIV-1 replication

It has been known for some time that HIV-1 infection, like many other viruses, causes an oxidative stress in the host cell which is accompanied by a massive ROS (reactive oxygen species) production. As an adaptation to these changes, the host-infected cell responds by activating antioxidant proteins. Amongst those, the selenoproteins are remarkably effective in red-ox reaction due to the presence of a selenium atom in their catalytic sites in the form of selenocysteine (also called the 21st amino acid). We have established a series of preliminary experiments showing that variations in selenium concentration modulate the replication of HIV-1 in T cells, concommitantly with the stimulation of selenoprotein expression. We will now concentrate on the interplay between activation of selenoproteins and HIV-1 replication in various cellular models.

(b) Isotopic fractionation of Zinc in the NCp7 of HIV-1

This is a novel exploratory and provocative project that we have started in collaboration with a team of geologists from the ENS with an expertise in isotopic fractionation (Dr. Balter, ENS de Lyon). Isotopic fractionation measures the ratio of stable isotopes in biological samples (tissues, organs, cells or even within metal-containing proteins). Interestingly, it was recently shown that a change in isotopic fractionation in tissues or organs can reflect pathological conditions.

The nucleocapsid protein (NCp7) of HIV-1 is an essential viral protein which contains 2 zinc finger motifs that are essentials for its biochemical function. Thus, it is possible that neo-produced HIV-1 viral particles concentrate either lighter or heavier Zn isotopes with consequences on both the biochemical activities of the enzyme and viral replication. We are currently investigating this hypothesis.

 

WP 3-Design of biotechnological tools

We have designed a novel in vitro cell free translation system based on the reticulocyte lysate which is supplemented with ribosomes isolated from different cells, tissues or even organs. With this unique combination, this cell-free system allows to study in vitro translation with different populations of ribosomes. This presents the advantage of studying the impact of ribosome modifications (by methylations or binding of specific factors) on in vitro transcribed mRNAs.

We have also engineered a new method to deliver the CRISPR/Cas9 editing tool by viral like particles that we called Nanoblades; this technique allows a rapid and efficient delivery to virtually any cells including primary cells. Nanoblades do not encode CAS9 but transfer a ready-made RNP immediately active in recipient cells. We will be improving the development of this technique and utilize it as a tool in many of our 'ongoing' projects.

 

   WP 4-RNA splicing and m6A modifications in Epstein Barr Virus (EBV) infected cells

EBV is a very large DNA virus with a size of 172 kbp and which codes for more than 90 proteins. It belongs to the human g-herpesvirus family and can cause many different cancers in immunocompetent individuals (adenocarcinoma, Burkitt lymphoma etc...) and lymphoma in immunodepressed patients. In vivo, EBV infects quiescent B cells and triggers a series of drastic changes in cellular gene expression, some of them at the post-trancriptional level. As such, they are mostly induced by the expression of 9 viral proteins, of which 6 are EBNAs (Epstein Barr Nuclear Antigens)  and 3 are LMPs (Latent Membrane Proteins), together with a large number of non-coding RNAs (both miRNAs and ncRNAs).

(a) EBV and alternative splicing

Upon EBV infection of primary B-lymphocytes, we will focus on the modification of the cellular and viral mRNA isoforms produced in infected cells. We have already performed an RNA-seq analysis of EBV-infected primary B cells at different time-points post-infection. Hence, we have identified many cellular mRNAs whose splicing pattern is modified early after infection. The objectives of our project are to characterize the full panel of mRNA isoform changes occurring at the beginning of EBV infection and determine how these patterns of expression can affect EBV-cell growth transformation.

(b) Epitranscriptomic in EBV infected cells

RNA N6‐adenosine methylation (m6A) was discovered 4 decades ago but the function of m6A and the characterisation of the cellular machinery that regulates its changes are only emerging. m6A is an abundant and reversible internal mRNA modification that mostly occurs at the vicinity of the stop codon in the 3'-UTR and in the coding region of the transcripts. These modifications were shown to have mutiple consequences on RNA metabolism at the level of transcript stability, poly(A) site selection and translational regulation. m6A methylation can also take place in the 5'-UTR of mRNAs where it was recently shown to exert an important role in the regulation of translation. We are looking at the epistranscriptomic pattern of EBV infected cells with the aim of determining the importance of m6A modifications on EBV replication and EBV induced cell transformation with a particular emphasis on the molecular mechanisms by which m6A modifications affect expression of viral and cellular transcripts.