Polymerase-tagged respiratory syncytial virus reveals a dynamic rearrangement of the ribonucleocapsid complex during infection.

Author summary Respiratory syncytial virus is a common respiratory disease among children under the age of five. Yearly, approximately three million children infected require hospitalization for treatment. However, no vaccine or effective antiviral treatment for respiratory syncytial virus yet exists. Understanding how respiratory syncytial virus replicates in cells will provide key knowledge for the development of more effective countermeasures. Here, we investigated the interactions between the large protein subunit of the respiratory syncytial virus polymerase, which is responsible for viral mRNA synthesis and replication, and other viral proteins during infection, as well as the localization of the large protein subunit over time in key viral structures. Our study provides new insights into the localization and activity of the respiratory syncytial virus polymerase, and suggests ribonucleocapsid complex rearrangement plays a key role in the function of the respiratory syncytial virus polymerase in replication of the virus, leading to a better understanding of respiratory syncytial virus biology. The ribonucleocapsid complex of respiratory syncytial virus (RSV) is responsible for both viral mRNA transcription and viral replication during infection, though little is known about how this dual function is achieved. Here, we report the use of a recombinant RSV virus with a FLAG-tagged large polymerase protein, L, to characterize and localize RSV ribonucleocapsid structures during the early and late stages of viral infection. Through proximity ligation assays and super-resolution microscopy, viral RNA and proteins in the ribonucleocapsid complex were revealed to dynamically rearrange over time, particularly between 6 and 8 hours post infection, suggesting a connection between the ribonucleocapsid structure and its function. The timing of ribonucleocapsid rearrangement corresponded with an increase in RSV genome RNA accumulation, indicating that this rearrangement is likely involved with the onset of RNA replication and secondary transcription. Additionally, early overexpression of RSV M2-2 fromin vitrotranscribed mRNA was shown to inhibit virus infection by rearranging the ribonucleocapsid complex. Collectively, these results detail a critical understanding into the localization and activity of RSV L and the ribonucleocapsid complex during RSV infection.