Abstract
Tobacco Mosaic virus (TMV) coat protein is well known for its ability to self-assemble into supramolecular nanoparticles, either as protein discs or as rods originating from the ~300 bp genomic RNA origin-of-assembly (OA). We have utilized TMV self-assembly characteristics to create a novel Flock House virus (FHV) RNA nanoparticle. FHV encodes a viral polymerase supporting autonomous replication of the FHV genome, which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. However, FHV viral genome size is strictly limited by native FHV capsid. To determine if this packaging restriction could be eliminated, FHV was adapted to express enhanced green fluorescent protein (GFP), to allow for monitoring of functional FHV RNA activity. Then TMV OA was introduced in six 3' insertion sites, with only site one supporting functional FHV GFP expression. To create nanoparticles, FHV GFP-OA modified genomic RNA was mixed in vitro with TMV coat protein and monitored for encapsidation by agarose electrophoresis and electron microscopy. The production of TMV-like rod shaped nanoparticles indicated that modified FHV RNA can be encapsidated by purified TMV coat protein by self-assembly. This is the first demonstration of replication-independent packaging of the FHV genome by protein self-assembly.
Highlights
Macromolecular assembly of virus capsid proteins has been used to create new formulations of virus-like particles (VLPs) and virus particles
One of the earliest studied self-assembly processes is that of Tobacco Mosaic virus (TMV); Fraenkel-Conrat et al first demonstrated that infectious TMV could be reconstituted in vitro from purified RNA and TMV coat protein under specific physiological conditions [1]
Arrow positions correspond to insertion sites that were engineered into the Flock House virus (FHV) RNA1 clone: C1- 3034 bp, C2- 3037 bp, C3- 2731 bp, C4- 3055 bp; (b) FHV-C2-green fluorescent protein (GFP)-P2A; (c) FHV-C4-GFP-ds where ds1 is
Summary
Macromolecular assembly of virus capsid proteins has been used to create new formulations of virus-like particles (VLPs) and virus particles. Further studies defined coat protein self-assembly properties [2], and characterization of a small RNA sequence within the TMV RNA that independently directs encapsidation [3] This sequence was used to direct encapsidation of non-native RNA content, of either a hybrid TMV RNA segment including a non-native 3' end [2], or a small gene coding sequence [4] that was successfully tested for co-translational protein expression. Expression of a reporter transgene in vitro suggested successful co-translational disassembly, and immunization and immune reactivity to the encapsidated transgene confirmed SFV function in vivo [5] These studies demonstrated that a novel virus composition could be created by TMV coat self assembly, there were limitations in the use of SFV encapsidated RNA, including a large
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