Journal: Communications Biology
Article Title: Cellular fate of a plant virus immunotherapy candidate
doi: 10.1038/s42003-024-06982-0
Figure Lengend Snippet: a CPMV structure: Cowpea mosaic virus (CPMV) has a bipartite ssRNA genome; the RNA-1 and RNA-2 are separately encapsulated into isometric 30 nm-sized capsids comprised of a small (S) and large (L) coat protein. CPMV RNA-1 encodes the 32 K protease cofactor, 58 K helicase, VPg, 24 K protease, and 87 K RNA-dependent RNA polymerase. RNA-2 encodes for the movement protein (MP), and the L and S coat proteins. PDB: 1NY7, CPMV structure generated with UCSF’s ChimeraX. b – j Nanoparticle characterization: b UV–VIS spectrum and the A260/280 ratio; intact CPMV have an A260/280 ratio of 1.7 ± 0.1. c , d DLS and AF4-DLS show a monodisperse ~30 nm-sized nanoparticle. e SEC shows the typical elution profile from a Superose 6 Increase column with a ~1.7 A260/280 ratio. f , g TEM of negatively stained CPMV and CryoEM of CPMV confirms the presence of monodisperse ~30 nm-sized CPMV. Scale bars at 50 nm and 100 nm, respectively. h Native gel visualized under UV light with RNA staining (GelRed) and white light after protein staining (Coomassie Blue); RNA and protein co-migrate, which indicates stable encapsulation of the RNA into the capsid. Denaturing gel: pure separations of CPMV L and S coat protein (L-CP and S-CP) at ~42 kDa and ~24 kDa, respectively. i Total RNA sequencing of RNA extracted from CPMV indicates 95% purity with sequences aligning with CPMV RNA-1 and RNA-2 [NC_003549.1 and NC_003550.1], 3% is unknown, and 0.4% aligns with black-eyed pea host. j Total protein sequencing of purified CPMV indicates 96.1% purity with sequences aligning with the CPMV coat proteins, 1.5% aligned with black-eyed pea host, and 2.4% aligned with a mixture of homo sapiens and bacterial contaminants. k Endotoxin detection from purified CPMV. N = 3.
Article Snippet: The AF4 system consisted of an isocratic pump (Agilent G1310A, Palo Alto, CA), well-plate autosampler (Agilent G1329A), AF4 separation channel (AF4, Wyatt Technology, Santa Barbara, CA), and a dynamic light scattering (Malvern Zetasizer Nano ZS) instrument.
Techniques: Virus, Generated, Staining, Encapsulation, RNA Sequencing, Sequencing, Purification
Waters Corporation manufactures this product 
![a CPMV structure: Cowpea mosaic virus (CPMV) has a bipartite ssRNA genome; the RNA-1 and RNA-2 are separately encapsulated into isometric 30 nm-sized capsids comprised of a small (S) and large (L) coat protein. CPMV RNA-1 encodes the 32 K protease cofactor, 58 K helicase, VPg, 24 K protease, and 87 K RNA-dependent RNA polymerase. RNA-2 encodes for the movement protein (MP), and the L and S coat proteins. PDB: 1NY7, CPMV structure generated with UCSF’s ChimeraX. b – j Nanoparticle characterization: b UV–VIS spectrum and the A260/280 ratio; intact CPMV have an A260/280 ratio of 1.7 ± 0.1. c , d DLS and <t>AF4-DLS</t> show a monodisperse ~30 nm-sized nanoparticle. e SEC shows the typical elution profile from a Superose 6 Increase column with a ~1.7 A260/280 ratio. f , g TEM of negatively stained CPMV and CryoEM of CPMV confirms the presence of monodisperse ~30 nm-sized CPMV. Scale bars at 50 nm and 100 nm, respectively. h Native gel visualized under UV light with RNA staining (GelRed) and white light after protein staining (Coomassie Blue); RNA and protein co-migrate, which indicates stable encapsulation of the RNA into the capsid. Denaturing gel: pure <t>separations</t> of CPMV L and S coat protein (L-CP and S-CP) at ~42 kDa and ~24 kDa, respectively. i Total RNA sequencing of RNA extracted from CPMV indicates 95% purity with sequences aligning with CPMV RNA-1 and RNA-2 [NC_003549.1 and NC_003550.1], 3% is unknown, and 0.4% aligns with black-eyed pea host. j Total protein sequencing of purified CPMV indicates 96.1% purity with sequences aligning with the CPMV coat proteins, 1.5% aligned with black-eyed pea host, and 2.4% aligned with a mixture of homo sapiens and bacterial contaminants. k Endotoxin detection from purified CPMV. N = 3.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9861/pmc11499861/pmc11499861__42003_2024_6982_Fig1_HTML.jpg)