2.  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.

3.  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.

4.  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).

5. 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.

6. RGD 44 Monomer

7. RGD 44 Trimer

13. Mutants Sub- cloning Agro infiltrated Capsid Extraction Successful Assembly RGD 44Completed Yes RGD 55Completed RGD 61Completed RGD 102Completed RGD 103Completed RGD 161Completed RGD 162Completed RGD 184Incomplete

16. 2x Low Speed Spins: 13G High Speed Spin: 50K RPM Low Speed Spin: 13G High Speed Spin: 50K RPM Resuspend Isolated Capsids 10X NaPO 4 Bentonite solution MgSO 4 RGD TYMV deveined plant tissue samples

17. Western blot Coomassie gel Ladder 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 27 kDa

18. Electron Microscopy Wild-type 28nm RGD 44 (60K) RGD 44 (125K) 50 nm

19.  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.

20. HHMI (Howard Hughes Medical Institute) URISC (Undergraduate Research, Innovation, Scholarship and Creativity) Dr. Kevin Ahern The Dreher Lab Special thanks to Josh Powell

21.  Kaur, G. / Valarmathi, M.T. / Potts, J.D. / Wang, Q., Biomaterials, 29 (30), p.4074-4081,Oct 2008