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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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In Vivo Delivery of Spherical and Cylindrical In Vitro Reconstituted Virus-like Particles Containing the Same Self-Amplifying mRNA
The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles(VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chloroticmottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying- mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.
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- PAR ID:
- 10523495
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- Molecular Pharmaceutics
- Volume:
- 21
- Issue:
- 6
- ISSN:
- 1543-8384
- Page Range / eLocation ID:
- 2727 to 2739
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acs.molpharmaceut.3c01105
