Abstract
Of the myriad viruses, very few have been shown to be capable of self-assembly in vitro from purified components into infectious virus particles. One of these is Cowpea Chlorotic Mottle Virus (CCMV), an unenveloped spherical plant virus whose capsid self-assembles around its RNA genome without a packaging signal. While heterologous RNA, not just cognate viral RNA, can be packaged into individual CCMV virus-like particles (VLPs), the RNA needs to fall within a certain range of lengths. If it is too short, it is packaged into particles smaller than wild type, or with two or more RNAs per capsid. If the RNA is too long, multiple capsids assemble around one RNA, and the RNA associated with these multiplet structures is not as RNase resistant. Further, as shown in the present work, 4200 nt appears to be the limiting length of RNA that can be packaged into single RNase-resistant CCMV VLPs. We explore the extent to which “overlong” RNA can be packaged more efficiently upon the addition of spermine, a polyvalent cation whose increasing concentration has been shown to compactify RNA. Finally, we show that the capsid protein of Brome Mosaic Virus (BMV), a bromovirus closely related to CCMV, also gives rise to multiplets when it is self-assembled with the same “overlong” RNA constructs, but with different distributions of multiplets.
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Acknowledgements
We are grateful to Jerrell Tisnado for expression and purification of recombinant capsid protein, and to all members of our research group for many helpful discussions about viral self-assembly over the past several years. This work has been supported financially by the National Science Foundation (Molecular and Cellular Biosciences Division, Genetic Mechanisms Program, Grants 1716975 and 2103700 to WMG).
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Duran-Meza, A.L., Chapman, A.G., Tanimoto, C.R., Knobler, C.M., Gelbart, W.M. (2023). The In Vitro Packaging of “Overlong” RNA by Spherical Virus-Like Particles. In: Comas-Garcia, M., Rosales-Mendoza, S. (eds) Physical Virology. Springer Series in Biophysics, vol 24. Springer, Cham. https://doi.org/10.1007/978-3-031-36815-8_3
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