4. As disease inducing agents - Known since 125 years
As useful/beneficial tools - Known since 50 years
4
Introduction
Plant viruses
5. Viruses as useful tools
1950’s
Bacteriophages as cloning vectors - Bacteriophage therapy
1970’s
Vaccines against virus diseases - Virus-like particles (VLPs)
1980’s
Plant viruses as expression vectors-
Pharmaceutical proteins in plants and plant cells.
2000’s
Viruses as nanocontainers - scaffolds or templates
(Khan and Tanveer, 2014)
5
6. • Template: A template is a pattern or mould which
fabricates structures same as their morphology and
topology.
• Biotemplate: Use of biological entities (DNA, RNA,
Protein cages and viruses) as a pattern for the synthesis
of other materials.
6
7. Why viruses?
o Small size
o Highly uniform structures
o Ability to self-assemble
o Ease of purification
7
8. General architectures of viruses
a) Non-enveloped virus
b) Non-enveloped virus with attachment proteins
c) Enveloped virus with glycoproteins playing the role of viral entry mediators.
(Stella et al., 2008)
8
10. Viruses and their capsid structure most utilized
as biotemplates
Cowpea mosaic virus
Cowpea chlorotic mottle virus 28-nm diameter
Brome mosaic virus 28-nm diameter
Tobacco mosaic virus 18 X 300 nm
28-nm diameter
10(Steinmetz and Evans, 2007)
11. Properties of virus as biotemplates for material synthesis
Remarkable plasticity
Dynamics during replication
Site specific delivery of cargo molecules
Genetic and chemical manipulation can impart new functions to
virus capsid architectures.
11(Stella et al., 2008) 11
12. Mechanism of genomic encapsidation
Three main ways for packaging of viral nucleic acid:
1. Requires ATP
Ex: ds DNA phages
(Aniagyei et al., 2010) 12
13. 2. Co-operative, simultaneous self-assembly of the nucleic
acid and its capsid.
Ex: Tobacco mosaic virus (TMV)
3. Pre-condensation of the nucleic acid followed by addition of
the surrounding protein subunits.
Ex: Brome mosaic virus (BMV), Cowpea chlorotic mottle
virus (CCMV).
13
(Aniagyei et al., 2010)
14. Approaches to non-genomic material
encapsidation
1. Synthesizing the cargo within the pre-assembled capsid
architectures.
Ex: CCMV, CPMV
(Stella et al., 2008) 14
15. Cont….
2. Assembling the protein cage around an existing cargo.
Ex: BMV, TMV
15
(Stella et al., 2008)
16. 16
3. Encapsidation by synthesis- covalent attachment of cargo
molecule to capsid protein
Ex: CPMV
Chemical coupling of therapeutic moieties to inner surface of capsid
(Stella et al., 2008)
17. Manipulation of coat protein subunits
Genetical
Chemical
Genetical and chemical
17
18. Genetic modification of viral capsid
Strategies for genetic manipulation:
Incorporation of artificial amino acids
By site directed mutagenesis
18
19. Modification of CCMV capsid interface
• Positively charged interior surface has been used for
nucleating inorganic mineralization
Example: synthesis of nanoparticles with anionic
polyoxometalate salts:
Tungstates
Molybdates
Vandates
(Young et al., 2008) 19
20. Cont…
Alteration from positive to negative charge through protein
design & genetic engineering.
Negatively charge density will be effective at directing the
surface nucleation of cationic transition metal oxides
• Fe2O3
• Fe3O4
(Young et al., 2008) 20
21. (a) Schematic for the application of the capsid interior as constrained
reaction vessels for material synthesis or encapsidation of a guest
nanoparticle.
(b) Transmission electron micrograph of magnetic Fe2O3 nanoparticles
mineralized within the CCMV capsid.
21(Young et al., 2008)
22. Ex: CCMV – Swelling mechanism / Gating
CCMV particles undergo reversible swelling, pH & metal ion dependent
structural transitions.
Results 10% increase in diameter.
Formation of 60 separate 2 nm sized openings.
Chemical modification of CCMV capsid interface
Cryoelectron microscopy and image reconstruction of the Cowpea chlorotic mottle virus. In an unswollen
condition induced by low pH (on the left), and in a swollen condition induced by high pH (on the right).
22
(Steinmetz and Evans, 2007)
23. Plant virus used as biotemplates and their application
Plant virus Application
Brome mosaic virus (BMV) Au nanoparticles .
ZnS semiconductor quantum dots ,
Iron oxide nanoparticles
Cowpea chlorotic mottle virus
(CCMV)
Enzyme nanoreactor, Biomedical applications,
Imaging agents
Cowpea mosaic virus (CPMV) Nanomaterials, Quantum dot decoration,
Biomedical applications, Drug delivery platforms
Tobacco mosaic virus (TMV) Nanomaterials, Surface modifications
Nanowires/mineralization
Turnip yellow mosaic virus
(TYMV)
Biomedical applications
Fluorescent labeling
Red clover necrotic mosaic virus
(RCNMV)
Au nanoparticles
Biomedical applications
23
25. 25
Objective: To use CPMV mutants as nanoscale scaffolds to
place gold nano particles.
(Blum et al., 2004)
26. CPMV mutants
26
The locations of inserted cysteines on the CPMV capsid .
(a) BC mutant, cysteines in pink. (b) EF mutant, cysteines
in white. (c) DM mutant, cysteines in white.
(Blum et al., 2004)
28. a) Unstained TEM image of gold
nanoparticles bound to an isolated
BC mutant CPMV virus.
b) Model of the BC mutant with one
gold nanoparticle bound per
cystine.
c) Unstained TEM image of gold
nanoparticles bound to an isolated
EF mutant CPMV virus
d) Model of the EF mutant with gold
particles bound to cystine with 7
nm interparticle distance.
e) Unstained TEM images of gold
nanoparticles bound to isolated DM
mutant of CPMV virus.
f) Model of DM mutant with 2nm
gold nanoparticle in each protein
subunit.
28
(Blum et al., 2004)
29. 29
Centre-to-centre distance of nearest neighbour gold nanoparticles
versus frequency for the EF mutant, the BC mutant and gold
nanoparticles alone.
(Blum et al., 2004)
30. 30
Objective: Synthesis of high surface area nanomaterials
using modified Tobacco mosaic Virus (TMV) templates.
(Royston et al., 2008)
31. Coat protein mutant of TMV
31
Structural location of the TMV-1Cys mutation.
(Royston et al., 2008)
32. Step1: TMV binds on the gold surface
Step2: it is activated with a Palladium
catalyst
Step3: it is finally coated with nickel
Schematic representation of the
TMV assembly and nickel or
cobalt coating process:
(Royston et al., 2008)
32
33. Images showing (a) a nickel-coated gold surface without TMV1cys, (b) a cobalt-coated
gold surface without TMV1cys, (c) a nickel-coated gold surface with TMV1cys and (d) a
cobalt-coated gold surface with TMV1cys
(Royston et al., 2008) 33
34. 34
FESEM images showing effects of concentration on assembly of TMV1cys
templates for nickel deposition. Concentration of
(a) o mg/ml TMV1cys (b) 0.01 mg/ml TMV1cys
(c) 0.1 mg/ml TMV1cys (d) 1 mg/ml TMV1cys
(Royston et al., 2008)
35. Objective: Synthesis of copper nanorods and nanowires using
TMV as a biotemplate
(Zhou et al., 2012) 35
36. Electroless deposition of Pd on wild type TMV
36
(a) TEM image of a Pd-TMV. (b) Higher resolution image from (a) white region
correspond to the grid and the dark area to the Pd-TMV. (c) SEM image of Pd-TMV
dispersed on Si wafer. (d) Picture of a centrifuge tube containing uniformly colored Pd-
TMV suspension.
(Zhou et al., 2012)
37. Electroless deposition of Cu on wild type TMV
• Mixing Pd-TMV into a Cu plating bath
• Cu plating bath CuSO4, EDTA and dimethylamine borane
(DMAB)
• After mixing, the mixture turned in to grey colour
• Reaction run for 12 minutes
• Collection of Cu-TMV rods were done by centrifugation of
samples after every three minutes and examined by TEM.
37
(Zhou et al., 2012)
38. Representative TEM images of Cu-TMV during time course experiment. The Cu-
TMV samples were taken from the plating bath (a) 3 min, (b) 6 min, (c) 9 min, and
(d) 12 min after the reaction started.
(Zhou et al., 2012) 38
39. (a)Schematic presentation of the synthesis of metallic nanowires
in the inner channel of TMV particle.
(b) TEM image of a single virion containing a 250 nm long nickel
wire inside the inner TMV channel
(Jing et al., 2012) 39
