Non-enveloped T=3 icosahedral symmetry of identical sequence.
Single stranded positive sense RNA virus.
Coat protein (CP) coded by sub-genomic strand.
180 coat proteins (CP), 60 as 1 of 3 quasi-equivalent sub-units.
60 A sub-units: pentameric capsomeres.
60 B sub-units and 60 C sub-units: hexameric capsomeres.
Arginine-glycine-aspartic acid (RGD) A tripeptide motif used by
Integrins as an attachment point.
RGD motifs are found within viral proteins and facilitate cellular adhesion.
Integrin (protein receptor) Integrin is a ligand used by some
cells and viruses for adhesion and cell signaling.
Stem cells up-regulate Integrin production.
Utilize RGD motif as a binding site for Integrin attachment. Multiple binding sites on exterior capsid. Regulate stem cell adhesion. Control stem cell growth and differentiation.
Mutant TYMV capsids are “vital for designing compatible biomaterials for tissue engineering purposes” (Gagandeep Kaur).
Arginine-Glycine-Aspartic acid (RGD) motifs can be
engineered on the exterior of Turnip yellow mosaic virus (TYMV) coat proteins (CP)
while maintaining the capsids: ability to assemble, structural stability, and allow binding of
Integrin to the RGD motifs.
RGD 44 Monomer
RGD 44 Trimer
MutantsSub-
cloning
Agro infiltrate
d
Capsid Extraction
Successful
Assembly
RGD 44Complete
dComplete
dCompleted Yes
RGD 55Complete
dComplete
dCompleted
RGD 61Complete
dComplete
d
RGD 102Complete
dComplete
d
RGD 103Complete
dComplete
dCompleted
RGD 161Complete
dComplete
d
RGD 162Complete
dComplete
dCompleted
RGD 184Incomple
te
2x Low Speed Spins: 13G
High Speed Spin: 50K RPM
Low Speed Spin: 13G
High Speed Spin: 50K RPM
Resuspend Isolated Capsids
10X NaPO4
Bentonite solution
MgSO4RGD TYMV deveined plant tissue samples
Western blot
Coomassie gelLadder Wild-type RGD 44 RGD 55 RGD 103 RGD 162
Coomassie gel prepared using:•Sodium dodecyl sulfate polyarcylamide gel electrophoresis (SDS-PAGE).•Coomassie Brilliant Dye.
Western blot prepared using:•SDS-PAGE.•Primary antibody: Rabbit anti-TYMV IgG.•Secondary antibody: Goat anti-rabbit IgG Horseradish Peroxidase (HRP) conjugate.
27 kDa
15 kDa
15 kDa
27 kDa
Electron Microscopy
Wild-type
28nm
RGD 44 (60K)
RGD 44 (125K)
50 nm 50 nm
RDG 44 expressed the mutant coat protein within the plant system, and self assembled forming capsids.
RDG 55, 103, or 162 coat proteins were not detected within the plant system. Future work will determine if the protein was or was not being expressed.
RDG 61, 62, 102, and 161 require further cloning techniques; transformation into Argobacterium, argoinfiltrated into the plant system, and capsid extraction to determine if coat protein can be expressed and self assemble within plant tissue.
HHMI (Howard Hughes Medical Institute) URISC (Undergraduate Research, Innovation, Scholarship and Creativity)
Dr. Kevin AhernThe Dreher Lab
Special thanks to Josh Powell
Kaur, G. / Valarmathi, M.T. / Potts, J.D. / Wang, Q. , Biomaterials, 29 (30), p.4074-4081,Oct 2008