This document summarizes research on using the virus-like particles (VLPs) of turnip yellow mosaic virus (TYMV) as a molecular container for multi-valent display and targeted drug delivery. Key points include: 1) TYMV VLPs were engineered to display "RGD" epitopes to target cancer cells overexpressing integrins and allow stem cell adhesion. 2) The stability of TYMV VLPs was enhanced by mutating residues in the coat protein, allowing reassembly from denatured states. 3) Biophysical studies characterized empty, virion and intermediate TYMV particles using techniques like gradient centrifugation and western blots. 4) Future

1. Engineer “Arg-Gly-Asp” (RGD)
epitopes for multi-valent display

2. RGD:
stem-cell
adhesion epitope
also target cancer
cells that up-regulate
integrin
TYMV with RGD substitution at residue 44

3. pH-dependent enhanced stability
Mutate internal scaffold N-terminus of TYMV CP
Wild-type 70°C
N-term mutant 70°C

4. Robust TYMV virus-
like particles in E. coli
In vitro reassembly
from a denatured
state

5. After many months
crystal structure
data for TYMV and
mutants!

6. TYMV can form a molecular “container” after
release of RNA, nano-inspired applications with
a protein shell with a small “hole”
before
After freeze-thaw

7. A CP cleavage product formed during RNA ejection
hindered in one of our “super-stable” virion mutants
&! SDS-PAGE: Coomassie stain ( ! Wt Mass Spec after freeze-
26
17 % RNA release
! C thaw: intact particle
!
"# $! !!ATC Virion ATC Virion / 01! / 23!
Wt Ky
&! SDS-PAGE: Coomassie stain (! ) *&) !!!*+, !!!&*, - , . , +!
Western blot
' ! 26 1 min.
/ 01!
%! exposure
Intensity
17
!
"#$! !!ATC Virion ATC%Virion
! / 01! / 23!
Wt Ky min.
10
exposure ) *&) !!!*+, !!!&*, - , . , +!
/ 23!
!
Western blot
'pH 11.5 pH 4 pH11.5 pH 1 min.
4
%! / 01!
exposure
Intensity
Ky Wt % min.
! +132
+132
10
# !Coomassie SDS-PAGE exposure / 23!
+132
pH 11.5 pH 4 pH11.5 pH 4
%!
13
% !
12 Ky 14Wt 15 16 Mass (Da) +132
+132
Fourier Transform-Ion Cyclotron Resonance
Wt CsCl gradient fractions
Coomassie SDS-PAGE
#! +132
(FTICR) Mass spectrometry

8. TYMV dual-subgenomic RNA for
expression of GFP and GUS.
Future downstream use for
vaccine development

9. Biophysical studies of TYMV
Characterization empty, virion and intermediate particles
Northerns, Westerns, gradient centrifugation, etc.

10. Previous Work (M.S degree at OHSU):
Signaling of a Cytomegalovirus chemokine
receptor US28: plenty of cloning, tissue
culture, immuno-precipitation/fluorescence
and construction of CMV knockout mutants
US28
chemokine

11. Recent TYMV article
& (cover image) Jan 2011,
more publications to come