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

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.

Towards a Quantitative Single Particle Characterization by Super Resolution Microscopy: From Virus Structures to Antivirals Design

“…Particularly, it was confirmed with correlative STED-FCS and by dSTORM that Gag plays a crucial role in Env cluster mobility at viral assembly sites, important for viral particle maturation (Roy et al, 2013;Chojnacki et al, 2017). On the other hand, Gag itself accumulates in budding areas forming three different patterns: small clusters (40-50 nm), medium clusters (140 nm) and patchy aggregates of bigger sizes (Figures 5B,C), characterized by dSTORM, PALM (Blanchard et al, 2020). (B) Expansion/light sheet of cells infected with live attenuated influenza NPs (Mascheroni et al, 2020).…”

“…(A) STORM imaging and distance measurement of RSV ribonucleocapsid proteins L (yellow) and N (magenta) at 6 and 8 h post infection (hpi). Scale bar 200 nm ( Blanchard et al, 2020 ). (B) Expansion/light sheet of cells infected with live attenuated influenza NPs ( Mascheroni et al, 2020 ).…”

“…Finally, the negative-sense RNA of the respiratory syncytial virus (RSV) needs the ribonucleocapsid of the virus to be protected in the cytoplasm, to be able to replicate and to translate. Using dSTORM it was possible to characterize the distribution of the proteins L and N of the ribonucleocapsid during the cycle of infection of RSV, understanding the roles of those proteins within the RNA of the virus and highlighting the rearrangement of the complex L-N during replication between 6 and 8 h post infection ( Blanchard et al, 2020 ) ( Figure 4A ).…”

EDP-938, a novel nucleoprotein inhibitor of respiratory syncytial virus, demonstrates potent antiviral activities in vitro and in a non-human primate model

“…Following entry into a cell, nucleocapsids are transported from the plasma membrane into the cytoplasm, where viral transcription/replication “factories” are established. These “factories” manifest initially as small granules containing N, P, L, M2-1 and viral RNAs, and increase in size over time to form inclusion bodies [ 34 , 35 ]. Studies with RSV have shown that these inclusions can be formed with just the N and P proteins, and are dynamic liquid organelles which can readily dissociate and re-associate [ 36 , 37 ].…”

Formation and Function of Liquid-Like Viral Factories in Negative-Sense Single-Stranded RNA Virus Infections

“…However, the role of RNA, in particular viral genomic RNA, in the phase separation remains to be explored. We also need to determine how a subcompartment is formed and maintained within IBs as well as the multiplicity and rearrangement of subcompartments possibly within an IB (e.g., see [ 108 ]). Virtually nothing is known about the host factors which regulate the assembly, maturation, and disassembly of IBs and their subcompartments.…”