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

• Published in: PLoS pathogens 2020-10, Vol.16 (10), p.e1008987-e1008987
• Main Authors: Blanchard, Emmeline L, Braun, Molly R, Lifland, Aaron W, Ludeke, Barbara, Noton, Sarah L, Vanover, Daryll, Zurla, Chiara, Fearns, Rachel, Santangelo, Philip J
• Format: Article
• Language: English
• Subjects: A549 Cells; Animals; Antibodies; Biology and Life Sciences; Biomedical engineering; Care and treatment; Chlorocebus aethiops; Development and progression; Engineering; Gene expression; Genetic aspects; Genetic research; Genomes; Health aspects; Humans; Infections; Localization; Medicine; Medicine and Health Sciences; Messenger RNA; Microscopy; Nucleocapsid Proteins - genetics; Nucleocapsid Proteins - metabolism; Proteins; Replication; Research and Analysis Methods; Respiratory syncytial virus; Respiratory syncytial virus infection; Respiratory Syncytial Virus Infections - genetics; Respiratory Syncytial Virus Infections - metabolism; Respiratory Syncytial Virus Infections - virology; Respiratory Syncytial Virus, Human - physiology; Ribonucleoproteins - genetics; Ribonucleoproteins - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; Structure-function relationships; Transcription; Transcription, Genetic; Vero Cells; Viral genetics; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Replication; Virus research; Viruses
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1008987

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 from in vitro transcribed 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.