Towards the elucidation of the morphogenesis, organization and functioning of respiratory syncytial virus factories – RSVFact

The respiratory syncytial virus (RSV) is a major and ubiquitous agent of respiratory viral infections in humans. It is responsible for more than 33 million cases/year of bronchiolitis in children worldwide and is also associated with a substantial disease burden in adults comparable to influenza, with most of the hospitalizations and mortality in the elderly. There are no vaccines or specific antivirals against RSV yet available. Although the development of a vaccine and specific antivirals is a high-priority public health objective, it is still impeded by our lack of knowledge of the molecular mechanisms of viral replication. RSV factories are cytoplasmic inclusions (IBs) in which replication and transcription of the genomic RNA occur, but these key structures still remain “black boxes”. Many unanswered questions regarding IBs organization and function remain: How does their structure support their key functions (regulation of viral RNA synthesis, encapsidation, mRNA and viral ribonucleoproteins sorting)? What are the mechanisms sustaining their biogenesis? What are the recruited cellular proteins?
Condensate formation by phase separation emerges as a new principle to explain the organization of living cells. Recently viral factories of few negative strand RNA viruses have been shown to exhibit properties of liquid organelles resulting of liquid liquid phase separation. Here, we aim at unravelling RSV factories biogenesis and progressing towards the understanding of their structure-dynamic-function relationships by addressing these questions in the frame of phase separation. To do so, we have gathered complementary and distinctive skills in our consortium. We will revisit the assembly and dynamics of RSV IBs by combining several model systems in an interdisciplinary approach: IBs in cells infected with recombinant viruses, pseudo-IBs resulting from transient expression of viral proteins, or IBs reconstituted from recombinant proteins in vitro. Taking advantage of a model of artificial IBs assembled in cells, we will identify cellular proteins recruited in IBs. As post-translational modifications (PMT) are known to control properties of condensates, we will identify PMT on viral proteins and investigate their impact on IBs properties and functions in our models. Our goals are to identify the elements controlling the assembly of IBs and to define the mechanisms involved in regulating IBs properties, dynamics, organization and function.
Elucidating the biogenesis, composition, and functioning of these key compartments in RSV multiplication will pave the way for the development of new antivirals for to treat RSV infections which represent a major public health issue.