RSV budding · Cellular and viral components in Respiratory Syncytial Virus (RSV) assembly and budding

Cellular and viral components in Respiratory Syncytial Virus (RSV) assembly and budding.

Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. Designing new antivirals requires detailed molecular and structural understanding of key steps in RSV replication. The project focused on the viral and cellular proteins involved in RSV budding, so far poorly understood process. One of the biggest gaps in our knowledge is RSV-host interaction network. The RSV Matrix (M) protein plays key roles in virus life cycle, coordinating viral assembly and budding at the plasma membrane, and it was suggested to recruit host factors essential for virus replication. We therefore screened for RSV M-host protein interactions using a high throughput microfluidics platform and a custom human proteome library as molecular bait. We identified 24 novel binding partners involved in a range of cellular pathways; host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodelling and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of M interaction with the actin-binding protein cofilin 1 and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV M in the host cell. Another aspect of the project focused on RSV M mutants defective in assembly and budding. We identified for the first time a key threonine residue within M that is critical for M phosphorylation and for infectivity of the released virus. We showed that single amino acid mutation impairs M distribution along viral filaments and modulates M oligomerization, causing disordered viral filaments formation and preventing infectious virus production. We also crystallized M in two crystal forms and show that it assembles into dimers. Dimerization interface mutants destabilized the M dimer in vitro. M dimerization mutants failed to assemble into viral filaments on the plasma membrane and budding and release of virus-like particles was prevented. Importantly, we show that M is biologically active as a dimer, and that the switch from M dimers to higher oligomers triggers viral filament assembly and virus production. Marie Curie Career Integration Grant has greatly increased my chances for integration as an independent group leader at European Institution. During the integration period I supervised and train students working with me on the project. I have regularly presented my work at UK and International meetings. This exposed me to the RSV as well as wider virology research community, expanded my network of collaborators, and enhanced my professional development. In summary, as Marie Curie Fellow I am now in an ideal position to be offered an independent group leader position in one of the European academic institutions.

Data: CORDIS, © European Union