How much genetic information in RNA form can be protected by a CCMV virus-like particle?
Ana Luisa Duran Meza, Sophie-Christine Porak, Abigail Chapman, Charles M. Knobler, William M. Gelbart
Abstract
The capsid protein of cowpea chlorotic mottle virus (CCMV) has been used extensively for in vitro packaging of heterologous RNA. The associated virus-like particles (VLPs) are spherical with a 3nm-thick/28nm-diameter protein shell and are therefore limited in the amount of RNA they can package. As shown in earlier work, when RNA lengths are longer than ~3500nt the RNA is no longer self-assembled exclusively into a single VLP.
Introduction
We are concerned here with in vitro reconstituted non-infectious virus-like particles (VLPs) that are self-assembled nucleocapsids formed from purified viral capsid protein and heterologous – non-viral – RNA. Essentially all VLP syntheses of this kind to date have been carried out with capsid protein from the plant viruses cowpea chlorotic mottle virus (CCMV) [1–5], brome mosaic virus (BMV) [6–9], and tobacco mosaic virus (TMV) [10–12], and from the bacterial virus MS2 [13–15].
Materials and Methods
RNA transcription and purification
Restriction enzymes from New England Biolabs (Ipswich, MA) were used as recommended by the manufacturer. The plasmids (sequences provided on request) were grown and purified using QIAprep (Qiagen DEU) following the manufacturer’s specifications. A Nodamura-RNA1-plus-ferritin-gene plasmid was linearized with NheI, SbfI, BsteII, AgeI and XbaI restriction enzymes to produce RNA transcripts with lengths of 3175, 3500, 3697, 3799 and 4026 nt, respectively.
Results and discussion
Incrementally larger RNAs ranging from 3175nt to 4433nt can be in vitro transcribed in comparable numbers
The purified in vitro transcribed RNAs were run in an agarose gel, as shown in Fig 1 in which lane 1 is an RNA ladder and the other lanes are RNA molecules of length 3175, 3234, 3500, 3697, 3799, 3970, 4026, 4197 and 4433 nt, with bands appearing at the corresponding positions.
Conclusion
In this work, RNAs of different lengths (3175, 3234, 3500, 3697, 3799, 3970, 4026, 4197, and 4433 nt) were encapsidated into VLPs using the plant virus CCMV capsid protein. We verified that not all of the RNAs packaged in the CCMV VLPs remain intact after exposure to the endonuclease RNase A. More explicitly, it was demonstrated that RNA up to a length of 3500 nt is fully protected by the CCMV VLP from RNaseA for RNase:RNA mass ratios up through 0.11:1 and that the VLPs containing longer RNA – which include multiplets of capsids – still give significant protection against RNase A.
Citation: Duran Meza AL, Porak S-C, Chapman A, Knobler CM, Gelbart WM (2025) How much genetic information in RNA form can be protected by a CCMV virus-like particle? PLoS One 20(12): e0336376.
https://doi.org/10.1371/journal.pone.0336376
Editor: Jian Xu, East China Normal University School of Life Sciences, CHINA
Received: March 21, 2025; Accepted: October 26, 2025; Published: December 10, 2025
Copyright: © 2025 Duran Meza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: W.M.G. acknowledges support from the National Science Foundation/Molecular and Cellular Biosciences Division (award number NSF MCB 2103700) and the National Institutes of Health/Institute of Allergy and Infectious Diseases (award number NIH NIAID R21 AI181743).
Competing interests: The authors have declared that no competing interests exist.
