Respiratory syncytial virus ribonucleoproteins hijack microtubule Rab11 dependent transport for intracellular trafficking

Respiratory syncytial virus (RSV) is the primary cause of severe respiratory infection in infants worldwide. Replication of RSV genomic RNA occurs in cytoplasmic inclusions generating viral ribonucleoprotein complexes (vRNPs). vRNPs then reach assembly and budding sites at the plasma membrane. However, mechanisms ensuring vRNPs transportation are unknown. We generated a recombinant RSV harboring fluorescent RNPs allowing us to visualize moving vRNPs in living infected cells and developed an automated imaging pipeline to characterize the movements of vRNPs at a high throughput. Automatic tracking of vRNPs revealed that around 10% of the RNPs exhibit fast and directed motion compatible with transport along the microtubules. Visualization of vRNPs moving along labeled microtubules and restriction of their movements by microtubule depolymerization further support microtubules involvement in vRNPs trafficking. Approximately 30% of vRNPs colocalize with Rab11a protein, a marker of the endosome recycling (ER) pathway and we observed vRNPs and Rab11-labeled vesicles moving together. Transient inhibition of Rab11a expression significantly reduces vRNPs movements demonstrating Rab11 involvement in RNPs trafficking. Finally, Rab11a is specifically immunoprecipitated with vRNPs in infected cells suggesting an interaction between Rab11 and the vRNPs. Altogether, our results strongly suggest that RSV RNPs move on microtubules by hijacking the ER pathway. Author summary Respiratory syncytial virus is the leading cause of severe lower respiratory infection in children worldwide and is increasingly recognized as a major respiratory pathogen in the elderly and frail. Yet, no curative treatment or vaccine is currently marketed. The late stages of RSV multiplication remain poorly understood despite they being potential targets for the development of antiviral strategies. In the infected cell, the viral genome is encapsidated and associated to the viral polymerase, to form the viral ribonucleoprotein (vRNP). The vRNPs are produced and assembled in cytoplasmic viral factories. The process ensuring their transport to the budding sites, at the plasma membrane, awaits to be precisely defined. Here we explored these mechanisms by tracking moving vRNPs in living infected cells. We developed an automated imaging pipeline allowing us to characterize the movements of vRNPs, at an unprecedented throughput. We then exploited the potential of our method to monitor the behaviour of the vRNPs during the infection. Using this approach, we document substantial trafficking of RSV along the microtubule network and demonstrate that RSV hijacks the recycling endosome pathway to promote the mobility of its vRNPs. Altogether, this work provides a cutting-edge approach allowing for live visualization of RSV RNP trafficking and critical data toward the understanding of RSV RNPs movements.

Automated summary

This study reveals that RSV RNPs move within cells by utilizing microtubules and the endosome recycling pathway, shedding light on the transportation mechanism of vRNPs.