In Vitro Conditions for Assembly of the Respiratory Syncytial Virus Nucleocapsid into Ring-like Oligomers or Helical Filaments Open Access
Raghavan, Anirudh (Spring 2022)
Abstract
A hallmark of the genomes of non-segmented negative-sense (-) RNA viruses is that the genomic RNA never exists in an isolated configuration – it is instead encapsidated by the nucleoprotein (N) into highly structured and ordered helical nucleocapsids (NCs), reminiscent of nucleosomes in eukaryotic cells. The NCs of the respiratory syncytial virus (RSV) can display multiple morphologies, including spherical, asymmetric, and filamentous conformations. Here, we analyze the morphology of recombinant nucleocapsid-like particles (NCLPs) of the respiratory syncytial virus assembled under different conditions. We examined the effects of modifying the integrated RNA length, and the effects of variations in buffer conditions, including salt concentration, ionic strength, pH, and the presence of reducing reagents under cryogenic conditions to assemble N into ring-like oligomers or higher-order helical filaments. We confirmed the conformational variability of the reconstituted RSV NCLPs and the constraints of the nucleocapsids upon binding to RNA using negative-stain and cryo-EM imaging. Our findings provide insights into N and RNA complexation into a loose oligomer form versus a tight helical skeleton form. We postulate that RNA length as well as flexibility of the C-terminal domain (CTD) arm of each N protomer, which can be manipulated in vitro via varying buffer conditions, affect RNA-binding as well as N oligomerization into higher-order structures. We hope that elucidation of NC assembly morphologies and identifying possible mechanisms leading to the formation of each can aid in future structural studies as well as help to develop future interventions to hinder efficient NC assembly, an essential prerequisite for RSV infection and proliferation.
Table of Contents
Introduction ............................................................................................................................................................................ 1
Figure 1 ........................................................................................................................................................................... 2
Figure 2 ........................................................................................................................................................................... 4
Figure 3 ........................................................................................................................................................................... 5
Figure 4 ........................................................................................................................................................................... 7
Materials & Methods ................................................................................................................................................................ 8
Results .................................................................................................................................................................................... 11
Figure 5 & 6 ..................................................................................................................................................................... 18
Figure 7 & 8 ..................................................................................................................................................................... 19
Figure 9 ........................................................................................................................................................................... 20
Figure 10 ......................................................................................................................................................................... 21
Figure 11 ......................................................................................................................................................................... 22
Discussion ............................................................................................................................................................................... 23
Figure 12 ......................................................................................................................................................................... 24
Figure 13 ......................................................................................................................................................................... 26
Figure 14 ......................................................................................................................................................................... 28
Supplemental Figures ............................................................................................................................................................... 29
Figure S1 ......................................................................................................................................................................... 29
Figure S2 ......................................................................................................................................................................... 30
Figure S3 & S4 ................................................................................................................................................................. 31
Figure S5 ......................................................................................................................................................................... 32
References ............................................................................................................................................................................... 33
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In Vitro Conditions for Assembly of the Respiratory Syncytial Virus Nucleocapsid into Ring-like Oligomers or Helical Filaments () | 2022-04-12 17:16:15 -0400 |
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