R2D2: Retroviral RNA Dynamics and Delivery

Retroviruses are RNA viruses with biogenesis and intracellular multiplication which leads to the release of viruses by budding on the cell. We studied two retroviruses: a oncogenic retrovirus: the Murine Leukemia Virus (MLV) that has been invaluable in our understanding of basic cellular processes (discovery of oncogenes, eukaryotic gene expression, viral pathology, and therapeutic trials with MuLV-based vectors) and the Human Immunodeficiency Virus of type 1 (HIV-1) that is the causative agent for the worldwide AIDS epidemic. Although the two viruses have different coding ability, they share a similar life-cycle in cultured cells.         

HIV-1                                                                                   MLV

Pictures from electron microscopy of free particles released from infected cells in culture 

All retroviruses contain a dimeric RNA genome formed from two identical monomers. After infection, this RNA is reverse transcribed into DNA for integration into the host chromosomes and thus perpetuating the viral infection. We are interested in the late steps of the retroviral life-cycle that start with the transcription of the integrated DNA. After transcription, a pool of the full-length transcript is spliced to produce subgenomic mRNAs encoding envelope, accessory and regulatory proteins. However, a subset of this full-length RNA pool is diverted from splicing machinery and exits the nucleus despite the lack of intron removal. This atypical export of unspliced RNA remains poorly understood, mainly for MLV. In the cytoplasm the full-length RNA has several destinies: it is captured by the translation machinery for Gag and Pol protein synthesis and/or it is targeted to the plasma membrane to be packaged in a dimeric state into assembling virions.

Different destinies of the retroviral RNA in the host cell

This particular metabolism of the retroviral RNA, crucial for the virus infectivity, still includes many shadowed areas. How is controlled the correct balance in the nucleus between RNA export and splicing and how is maintained the equilibrium between RNA translation and RNA packaging in the cytoplasm? While the genomic RNA is in large minority among the other cellular and viral RNAs, how is it specifically selected to be packaged as dimer into the new particles? We know that Gag polyprotein is a close partner of the RNA during these late phases. We study how Gag via its nucleocapsid domain (GagNC) specifically binds the RNA and mediates its dimerization and encapsidation into new viral particles. How the viral components Gag, GagPol, and how RNA reaches the virus assembly sites at the cell surface remains to be elucidated.

The team is interested in the molecular and structural mechanisms and the cell biological aspects involved in the viral RNA metabolism. In recent years, our efforts have been focused on the dynamic aspect of these mechanisms (as shown in figure below). For this purpose, we develop advanced and highly sensitive approaches to conduct real-time investigations in living cells at the level of the single cells and at the scale of unique RNA molecules.

We hope that the study of the molecular mechanisms of virus biogenesis and replication will help to fight retrovirus-induced disease, including AIDS and cancer.  

Vidéo made with TIRF microscopy showing the formation of fluorescent HIV-1 particles (white dots) on the surface of the host cells. The viral productions are very different among the four cells observed.