Ubiquitination and de-ubiquitination events in the regulation of viral replication – Ubi-or-not-Ubi

Cells are tightly regulated by sophisticated processes. Among them, post-translational modifications of proteins is essential. Recently, the covalent modification of proteins by ubiquitin and degradation by the proteasome has emerged as a major, critical process by which virtually all aspects of cell biology are regulated. Given this importance, it is therefore not surprising that many pathogens, including viruses, were found to exploit and/or interfere with this regulatory pathway.

The proposal "Ubi-or-not-Ubi : ubiquitination and de-ubiquitination events in the regulation of viral replication" aims at the elucidation of novel molecular and cellular mechanisms underlying host/virus interactions during viral RNA replication.

This project is based on recent results that were acquired on the model plant RNA virus TYMV, in the course of the ANR Blanc project "Phospho-Pol" funded in 2006. First, we obtained evidence that a key viral replication protein - the polymerase, is targeted by the Ubiquitin-Proteasome system in the course of the viral infection process. Second, we observed that another viral replication protein - named 98K, is capable of inhibiting this degradation process. Through detailed biochemical analyses, we could demonstrate that 98K displays a deubiquitinase activity. Strikingly, the viral polymerase was confirmed to be a substrate of this enzyme, and the deubiquitinase activity was found critical for viral infectivity. These findings, which to our knowledge are without any precedent, clarified some aspects of the viral replication process and its regulation, but they also opened new (and exciting !) questions, and the aim of the proposed project is to answer some of them.

In the proposed project "Ubi-or-not-Ubi", we will further characterize the reversible ubiquitination events occurring during viral infection, using a combination of different in vivo, in vitro and in silico approaches. We will make use of the recent knowledge acquired on the TYMV polymerase and the TYMV-encoded deubiquitinase to further investigate the ubiquitination of the viral polymerase, with the aim of characterizing the host ubiquitin ligase involved in this process. In addition, a great deal of our efforts will be directed towards the study of the newly discovered viral deubiquitinase. Apart from the characterization of its structure / function relationships, our objective is to identify novel protein substrates of this viral enzyme, and to analyze the global changes resulting from its expression. Finally, we aim to integrate these findings to clarify the contribution of ubiquitination and deubiquitination events to the efficiency of viral replication.

This basic research project will be achieved by a collaborative effort between three academic laboratories with complementary expertise: Partner n°1 (Equipe de Virologie Moléculaire, Institut Jacques-Monod, Paris, driven by Dr. Isabelle Jupin) is specialized in molecular and cellular virology; Partner n°2 (Equipe de Radiocristallographie, Laboratoire de Virologie Moléculaire et Structurale, CNRS, Gif sur Yvette, driven by Dr. Stéphane Bressanelli) is specialized in structural biology, and Partner n°3 (Equipe de Modélisation en Biologie intégrative, Institut Jacques-Monod, Paris, driven by Prof. Khashayar Pakdaman) is specialized in systems biology.

The proposed project should generate results at the leading front of research on the involvement of the ubiquitin proteasome system in host / pathogens interactions, which the international community is beginning to investigate. Understanding the molecular dialogue between viruses and host cells is fundamental, and opens up fascinating new areas for research that span from basic cell biology to therapeutic interventions against viruses.