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