Nanoparticle encapsidation of Flock house virus by auto assembly of Tobacco mosaic virus coat protein

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.

Figure 1

Schematic representation of the Flock House virus RNA1 vector constructs. (a) FHV RNA1 genome with subgenomic proteins, B1 and B2. Arrow positions correspondto insertion sites that were engineered into the FHV RNA1 clone: C1- 3034 bp, C2- 3037 bp, C3- 2731 bp, C4- 3055 bp; (b) FHV-C2-GFP-P2A; (c) FHV-C4-GFP-ds where ds1 is 2518–3055 bp and ds2 is 2518–3107 bp of FHV RNA1 were cloned into FHV-C4-GFP respectively; and (d) FHV-C4-GFP-RNA2 where 509–868 bp from FHV RNA2 was cloned into FHV-C4-GFP.

Figure 2

Fluorescence images of FHV RNA1 GFP plasmid constructs. BHK-21 cells were transfected with 2 µg of in vitro transcribed FHV RNA with DMRIE-C transfection reagent. Transfected cells were incubated at 37 °C for 4 h after which fresh growth media was added and cells transferred to 28 °C. Images were taken at 48 h post-transfection with a Nikon Eclipse TS-100 microscope and NIS Elements BR 4.11.00 imaging software. All images are at a 400× magnification. (a) FHV-C1-GFP; (b) FHV-C2-GFP; (c) FHV-C3-GFP; (d) FHV-C4-GFP; (e) FHV-C1-GFP-P2A; (f) FHV-C2-GFP-P2A; (g) FHV-C4-GFP-P2A; (h) FHV-C4-GFP-ds1; (i) FHV-C4-GFP-ds2; (j) FHV-C4-GFP-RNA2; (k) SFV-GFP-OA; and (l) non-transfected cells.

Figure 3

Time course assay observing daily fluorescence of different FHV GFP clones over a period of 120 h. BHK-21 cells were exposed to 2 µg of FHV RNA via liposome-mediated transfection, incubated at 28 °C and observed for fluorescence daily. All cell images are at a 400× magnification: (a) FHV-C2-GFP; (b) FHV-C1-GFP-P2A; (c) FHV-C2-GFP-P2A; and (d) FHV-C4-GFP-P2A and SFV-GFP. Images from expression vectors that had undetectable to low levels of fluorescence were not included in this figure.

Figure 4

Western blot analysis demonstrating P2A cleavage efficiency in BHK-21 cells at 48 h. Cell lysates were collected at 48 h after confirmation of fluorescence and 20 µL of cell lysate was loaded onto 8%–16% Criterion™ TGX™ precast gel. The gel was transferred to a PVDF blot, and the blot was blocked and incubated with a monoclonal rabbit anti-GFP antibody and secondary incubation was with a goat-HRP-anti-rabbit antibody. Blots were developed with a HRP development kit after an hour and exposed on X-ray film. (a) FHV-C1-GFP; (b) FHV-C1-GFP-P2A; (c) FHV-C2-GFP; (d) FHV-C2-GFP-P2A; (e) FHV-C4-GFP; (f) FHV-C4-GFP-P2A; (g) DMRIE-C only; (h) un-transfected cells; and (i) eGFP protein 0.1 µg.

Figure 5

Construction and characterization of FHV-OA clones. (a) Schematic diagram showing the 6 OA insertion sites; (b) Fluorescence images of FHV-GFP-OA clones that were taken at 48 h after liposome-mediated transfection of BHK-21 cells; and (c) Time course assay showing GFP expression kinetics of FHV-C2-GFP-OA in BHK-21 cells after transfection with 2 µg of FHV-C2-GFP-OA RNA. All cell images are at 400× magnification.

Figure 6

Characterization of in vitro encapsidated products by comparing migration rates on a Coomassie-stained 0.5% w/v agarose gel. TMV CP was mixed with OA-containing RNAs in the presence of a 25 mM phosphate buffer and incubated overnight at room temperature. The resulting mixture was polyethelene glycol (PEG) precipitated, pelleted and resuspended with 50 µL nuclease free PBS. 20 µg of encapsidated product was loaded onto a 0.5% Tris-phosphate-EDTA (TPE) gel and electrophoresis was performed at 300 mA for 3 h. The gel was stained with Coomassie blue overnight and de-stained for 2 days. TMV wild type virus and TMV CP were run as a control and size markers where the TMV WT monomer runs at 6 kb and dimerruns at 12 kb: (a) TMV WT 20 µg; (b) TMV WT 10 µg; (c) TMV CP 5 µg; (d) TMV CP 10 µg; (e) TMV CP 20 µg; (f) in vitro encapsidated wt TMV RNA; (g/h) in vitro encapsidated FHV-C2-GFP-OA, two independent encapsidations; and (i) In vitro encapsidated SFV-β-Gal-OA was also run as a positive control for successful encapsidation.

Figure 7

Electron micrographs of in vitro encapsidated (IVE) OA-containing RNA with TMV CP. Grids were coated with 100–200 µg/mL of in vitro encapsidated particles, negatively stained with 1% phosphotungstic acid (PTA), air dried, observed with a Philips CM120 microscope and imaged with Gatan MegaScan 795 digital camera. Black bar represents 100 nm: (a) Wild type TMV virions approximately 300 nm in length; (b) TMV RNA encapsidated with TMV coat proteins are approximately 300 nm in length; (c) FHV-C2-GFP-OA RNA encapsidated with TMV coat proteins approximately 187nm in length; and (d) SFV-GFP-OA RNA encapsidated with TMV coat proteins approximately 600nm in length.