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

• Type of Material: Article
• Authors: Emmeline L. Blanchard, Georgia Institute of Technology; Molly R. Braun, Boston University; Aaron W. Lifland, Georgia Institute of Technology; Barbara Ludeke, Boston University; Sarah L. Noton, Boston University; Daryll Vanover, Georgia Institute of Technology; Chiara Zurla, Georgia Institute of Technology; Rachel Fearns, Boston University; Philip Santangelo, Emory University
• Language: English
• Date: 2020-10-01
• Publisher: PUBLIC LIBRARY SCIENCE
• Publication Version: Final Published Version
• Copyright Statement: © 2020 Blanchard et al
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.ppat.1008987
• Title of Journal or Parent Work: PLOS PATHOGENS
• Volume: 16
• Issue: 10
• Start Page: e1008987
• End Page: e1008987
• Grant/Funding Information: This work was supported by the National Institutes of Health R01AI113321 and R01GM114561, (www.nih.gov). ELB was supported by National Science Foundation Graduate Research Fellowship Program [Grant No. DGE-1650044] (www.nsf.gov) and the National Institutes of Health GT Biomat Training Grant [T32-EB006343] (www.nih.gov).
• Keywords: NUCLEOPROTEIN; GENE-EXPRESSION; MESSENGER-RNA; Virology; VESICULAR STOMATITIS-VIRUS; INCLUSION-BODIES; Science & Technology; N-PROTEIN; Life Sciences & Biomedicine; OPEN READING FRAME; M2-2 PROTEIN; RNA REPLICATION; Microbiology; TRANSCRIPTION ELONGATION-FACTOR; Parasitology
• Research Categories: Biology, Genetics; Biology, Virology; Biology, Parasitology

Abstract

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.