Optimal Conditions for In Vitro Assembly of Respiratory Syncytial Virus Nucleocapsid-like Particles

• Type of Material: Article
• Authors: Yunrong Gao, Emory University; Anirudh Raghavan, Emory University; Bowei Deng, Emory University; Jae Lee, Emory University; Bo Liang, Emory University
• Language: English
• Date: 2023-02-01
• Publisher: MDPI
• Publication Version: Final Published Version
• Copyright Statement: © 2023 by the authors. Licensee MDPI, Basel, Switzerland.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.3390/v15020344
• Title of Journal or Parent Work: Viruses
• Volume: 15
• Issue: 2
• Grant/Funding Information: This research was funded by the National Institute of Health, grant number R01GM130950 and the University Research Committee (URC) Award, Emory University.
• Keywords: respiratory syncytial virus (RSV); pH; ionic strength; Antiviral Agents; Adenine; Respiratory Syncytial Virus, Human; Nucleocapsid; Sodium Chloride; nucleocapsid-like particles (NCLPs); salt concentration; nucleocapsid (NC); negative stain EM; Virion; RNA
• Research Categories: Biology, Genetics

Abstract

The nucleocapsids (NCs) of the respiratory syncytial virus (RSV) can display multiple morphologies in vivo, including spherical, asymmetric, and filamentous conformations. Obtaining homogeneous ring-like oligomers in vitro is significant since they structurally represent one turn of the characteristic RSV NC helical filament. Here, we analyzed and optimized conditions for forming homogenous, recombinant nucleocapsid-like particles (NCLPs) of RSV in vitro. We examined the effects of modifying the integrated RNA length and sequence, altering incubation time, and varying buffer parameters, including salt concentration and pH, on ring-like NCLPs assembly using negative stain electron microscopy (EM) imaging. We showed that high-quality, homogeneous particles are assembled when incubating short, adenine-rich RNA sequences with RNA-free N associated with P (N0P). Further, we reported that a co-incubation duration greater than 3 days, a NaCl concentration between 100 mM and 200 mM, and a pH between 7 and 8 are optimal for N-RNA ring assembly with polyadenine RNA sequences. We believe assembling high-quality, homogeneous NCLPs in vitro will allow for further analysis of RSV RNA synthesis. This work may also lend insights into obtaining high-resolution nucleocapsid homogeneous structures for in vitro analysis of antiviral drug candidates against RSV and related viruses.