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Viruses and Virus-Like Protein Assemblies——Chemically
Programmable Nanoscale Building Blocks
L. Andrew Lee, Zhongwei Niu, and Qian Wang
Department of Chemistry & Biochemistry and Nanocenter, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA
This paper is dedicated to Professor Yu-Fen Zhao on the occasion of her 60th birthday.
Received: 7 January 2009 / Revised: 15 February 2009 / Accepted: 16 February 2009 ©Tsinghua University Press and Springer-Verlag 2009. This article is published with open access at Springerlink.com
DOI 10.1007/s12274-009-9033-
Review Article
Address correspondence to wang@mail.chem.sc.edu
ABSTRACT
Supramolecular proteins are generated using a limited set of twenty amino acids, but have distinctive
functionalities which arise from the sequential arrangement of amino acids configured to exquisite three-
dimensional structures. Viruses, virus-like particles, ferritins, enzyme complexes, cellular micro-compartments,
and other supramolecular protein assemblies exemplify these systems, with their precise arrangements of tens
to hundreds of molecules into highly organized scaffolds for nucleic acid packaging, metal storage, catalysis
or sequestering reactions at the nanometer scale. These versatile protein systems, dubbed as bionanoparticles
(BNPs), have attracted materials scientists to seek new opportunities with these pre-fabricated templates in a
wide range of nanotechnology-related applications. Here, we focus on some of the key modification strategies
that have been utilized, ranging from basic protein conjugation techniques to more novel strategies, to expand
the functionalities of these multimeric protein assemblies. Ultimately, in combination with molecular cloning
and sophisticated chemistries, these BNPs are being incorporated into many applications ranging from
functional materials to novel biomedical drug designs.
KEYWORDS
Bionanoparticles, virus, bioconjugation, nanomaterials, bioimaging, drug delivery
Introduction
Biological materials have been extensively utilized as
starting precursors to generate ornate nanostructures
for materials development [1 7]. For example, the
finite number of building blocks and established base-
pairing rules in nucleic acids provides a predictable
system that has been used to design two- and three-
dimensional lattices [8 10], geometrical shapes [11,
12], and other nanopatterns [13, 14]. Nature also
employs protein assemblies, such as viruses and
virus-like particles (VLPs) [15], ferritins [16, 17], heat
shock protein cages [18], and enzyme complexes [
21] to form robust biosynthetic machineries (Fig. 1).
These protein shells, or bionanoparticles (BNPs), are
highly organized nanoscale materials with robust
chemical and physical properties while still being
capable of modification by genetic and chemical
methods. A myriad of viruses and VLPs have been
genetically and chemically reprogrammed to function
as drug/gene delivery vehicles [22 24], vaccines [
27], and nanomaterials [28 34].
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In comparison to conventional synthetic particles,
viruses and viral-like protein assemblies are
attractive for the development of new materials for
the following reasons:
1) They represent very stable and beautiful self-
assembled architectures at the nanometer level
with sizes ranging from 10 nm to 200 nm, which
are otherwise very difficult to make by standard
synthetic methods in the laboratory.
( 2 ) T h re e - d i m e n s i o n a l s t r u c t u re s c a n b e
characterized at near atomic resolution.
(3) The composition and surface properties of the
viruses can be controlled using molecular biology.
(4) They can be purified inexpensively on a large
scale, a crucial advantage when considered for
materials development.
(5) For each type of virus and virus-like protein
assembly, all the particles are identical. We can
therefore envision them as truly mono-disperse
nanoparticles.
Recently, there have been quite a few detailed
review articles which summarize the application
of viruses or viral protein cages in biomedicine
and materials science [35 39]. In this review, we
highlight some of the general approaches utilized
to modify these viruses, ferritins, and other protein
assemblies in order to impart novel functionalities for
nanotechnology applications.
1.1 Chemically addressable bionanoparticles
Many of the basic protein conjugation schemes
permeate fundamental BNP chemistry, targeting
endogenous amino acids, such as lysines, glutamic
or aspartic acids, and cysteines. Less commonly
targeted functional groups, such as the phenol ring
of tyrosines, have also been incorporated into this
approach (Fig. 2). The systematic characterization
of cowpea mosaic virus (CPMV) [40 44], along with
studies of cowpea chlorotic mottle virus (CCMV)
[45], bacteriophage MS2 [46], heat shock protein [18],
tobacco mosaic virus (TMV) [47], and turnip yellow
mosaic virus (TYMV) [48], have shed light on the
unique chemical reactivities and physical properties
of these individual BNPs. These initial discoveries
have been integred to design BNPs as nano-scaffolds
for drug delivery, bioimaging, biomineralization, and
even tissue engineering.
For instance, based on the crystal structure of
CPMV (Fig. 3(a)) [49], only a few lysine residues
p e r a s y m m e t r i c u n i t a p p e a r e x p o s e d t o t h e
solvent (Fig. 3(b)), whereas the majority of the
lysine groups are found buried or interacting with
neighboring residues (Fig. 3(c)). Studies indicated
that reactions with N -hydroxysuccinimide (NHS)
ester-functionalized fluorescein dyes preferentially
targeted these surface-exposed lysines [41, 50].
Figure 1 Three-dimensional structures of bionanoparticles: moving clockwise from the top are ferritin, heat shock protein, lumazine synthase, brome mosaic virus, MS2 bacteriophage, cowpea chlorotic mosaic virus, turnip yellow mosaic virus, cowpea mosaic virus and in the center, tobacco mosaic virus. Models were generated using PyMol (www.pymol.org) with coordinates obtained from the RCSB Protein Data Bank (www.pdb.org)
Figure 2 Conventional protein conjugation strategies targeting the endogenous amino acids (lysine, cysteine, aspartic or glutamic acids, and tyrosine) on bionanoparticles
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excellent modification of tyrosine residues located
inside the bacteriophage MS2, enhancing its use
for storage of drugs and as an MRI contrast agent
[57 59]. This protocol has been expanded with a
larger pool of reagents and reaction conditions for
TMV, demonstrating this method to be a flexible
and orthogonal procedure for BNP modification at
tyrosines (Fig. 4).
1.2 Chemical modification in combination with
genetic mutation
Surprisingly, no reactive cysteine residues are
exposed on the exterior surface of most BNPs, a
reasonable assumption being that evolution has
disfavored particles forming inter-particle cross-
links via disulfide bonds. This presents the unique
opportunity to genetically position the cysteine
residue on strategic locations of viruses and protein
shells, after which the sulfhydryl group can be
selectively targeted with thiol-selective reagents. For
instance, the virus coat protein of CPMV expresses
14 cysteine residues (3 in the small subunit and 11
in the large subunit); however, treatment of wild-
type CPMV with thiol-selective reagents yielded
little or no attachments to the virus [42, 44]. Lin and
collaborators have engineered the cysteine residue on
surface-exposed loops based on several design criteria
[60]. The mutants expressed the cysteine residue as
part of an added small loop or as a point mutation
(Fig. 5(a)), resulting in 60 copies of the inserted thiol
being displayed symmetrically around the 30-nm-
diameter particle (Fig. 5(b)). Compared to the native
virus, the new inserted cysteines demonstrated
higher reactivity, with nearly all of the inserted
thiol groups being chemically modified at very low
concentration of a maleimide electrophile at neutral
pH (Fig. 5(c)) [40]. Biologically-relevant proteins
(T4 lysozyme, Her2, and LRR domain of internalin)
could be anchored through the use of a bifunctional
linker to the surface of an icosahedral virus via the
engineered cysteine residue, meanwhile retaining the
structural and biological functionalities of the virus
and its conjugates [61]. The innate structural features
of the virus have also been exploited to pattern
gold nanoparticles around the three-dimensional
space [62]. In another system, the heat shock protein
from Methanococcus jannaschii (MjHsp) has been
engineered with a cysteine residue housed within
the interior and by coupling the reactivity of the
cysteine with a pH-sensitive maleimide derivative,
an antitumor drug was linked to the interior surface
and selectively released upon decrease in pH [63].
Culver et al. designed and functionalized cysteine
substituted-TMV particles with fluorescent dyes,
and the modified TMV particles were then partially
disassembled to expose the single-stranded viral
Figure 4 Scheme of tyrosine modifi cation in bacteriophage MS2 using various diazonium salts
RNA. The exposed ssRNA strand was then utilized
to hybridize to complementary DNA sequences
patterned on surfaces [64, 65]. Francis and co-workers
expressed TMV coat protein in a bacterial system
to generate cysteine substituted-TMV coat proteins,
which were modified with fluorescent chromophores
for the purpose of generating a light-harvesting
system. By controlling the pH and ionic strength, the
proteins self-assembled into long fibrous structures
which were capable of positioning the chromophores
for efficient energy transfer [66].
These studies highlight an important feature of
BNPs, namely that chemically reactive groups can
be genetically engineered to selectively position
drug molecules, imaging agents, and biologically
relevant molecules on the three-dimensional
template, which is extremely difficult to realize using
synthetic nanoparticles. In particular, the regio-
precision of surface functionalization can be crucial
for many biomedical applications. For example,
in their recent endeavor, the Finn group exploited
the architectural features of the virus to explore
how spatial distribution and polyvalent display
of antigenic carbohydrates would modulate the
mammalian immune response [67]. Moreover, a new
way to enhance carbohydrate immunogenicity has
been reported by means of ordered display on the
surface of the CPMV capsid [68, 69]. In another study,
murine polyoma VLPs genetically engineered with
eight glutamic acids and one cysteine residue in one
of its exterior loops tethered the anti-tumor antibody
B3 via electrostatic interactions [70]. The antibody
fragment was modified at the C -terminus with a
peptide sequence (Arg 8 CysPro), which associates
with the mutant VLP, and the complex is further
stabilized by the formation of a disulfide linkage. This
coupling reaction yielded polyoma VLPs with 30 40
antibody fragments bound to the surface, allowing
the modified VLPs to bind to breast carcinoma cells
with high efficiencies [70].
1.3 Novel bioconjugation techniques
Not all proteins are created equal, ergo all BNP
scaffolds do not exhibit similar reactivities or
selectivity. The need for selective reactions in complex
biomolecules such as BNPs creates a new challenge
to design alternative schemes to covalently modify
proteins in aqueous or physiological solutions,
while retaining their original structural integrity
and functionality. Reactions such as Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) [71] and other
examples of transition metal catalysis [72] have
addressed this need by successfully demonstrating
cell-surface labeling and virus modifications.
The CuAAC reaction, a member of the family of
click reactions, has emerged as an excellent tool to
Figure 5 (a) Cysteine residues engineered on CPMV on the protruding loop ( B C), shown here in red, which have a tendency to aggregate without the presence of reducing agents. The lesser exposed loop ( E F), shown in green, is more resistant to aggregation. (b) Each mutation resulted in 60 reactive thiol groups positioned around a predictable 3-D space, with a 28 Å spacing between each thiol group on the B C (red) loop versus a 50 Å spacing on the E F (green) loop. (c) The electron density difference after modification with gold nanoparticles, shown in gold, clearly shows the attachment site is at the cysteine. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reprinted with permission
(a) (b) (c)
agents, cell targeting, and therapeutic drugs to reduce
non-specific cargo release and side-effects in patients.
Combinations of the aforementioned bioconjugation
strategies have been utilized to afford additional
handles on BNPs, which should eventually lead to
such multi-functional constructs. Therefore, both
signaling moieties (like fluorescent or magnetic
molecules) and biological recognition motifs (such
as antibodies, oligonucleotides, ligands, receptors,
or chemical sensors) can be simultaneously attached
to BNPs, leading to possible nanosized vehicles for
biosensing or drug delivery.
For example, a CPMV mutant, expressing the
uniquely reactive cysteine residue between residues
G98 and K99 of the large subunit, still possesses all of
the natural chemical reactivity of the exposed lysine,
K38 [43, 50]. Therefore, one can sequentially modify
the lysines with an NHS-ester (or isothiocyante)
functionalized group and the cysteines with a
maleimide group [43]. In one case, the CPMV
mutant was functionalized with fluorescent dyes
and immunoglobulin (chicken or mouse IgGs) to
test its potential use in immunoassays (Fig. 7(a))
[83]. It has also been shown that fluorescent dyes can
be anchored on CPMV with controlled separation,
which prevented the formation of non-fluorescent
dimers and subsequent quenching, and thus afforded
highly fluorescent viral nanoparticles [84]. Such kind
Figure 7 (a) Bifunctional CPMV displaying fluorescent dye and immunoglobulin for immunoassays; (b) integrin targeting peptide sequence (Arg-Gly-Asp) engineered on a heat shock protein as a potential drug delivery agent to target melanomas; (c) terbium ligand complex conjugated to TYMV to generate time-resolved fl uoroimmuno assays
(a) (b)
(c)
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of engineered viral particles can be used as probes in
microarray-based genotyping assays and sandwich
immunoassays with improved sensitivities [83, 84].
Francis and co-workers generated doubly
labeled TMV and MS2 by utilizing intrinsic amino
acids to functionalize the exterior and the interior
with small molecules, dyes, and polymers [47, 57].
Douglas, Young, and co-workers generated MjHsp
with cysteines engineered in the interior of the shell
and the cell targeting moieties on the exterior to
direct the protein shell to melanomas (Fig. 7(b)) [85].
Wang and group recently generated TYMV labeled
with a terbium complex and biotin as a prototype
BNP for time-resolved fluoroimmuno assays (Fig.
7(c)) [48], whilst Culver and co-workers have
recently developed an elegant approach to assemble
multifunctional TMV onto a solid substrate [65].
In addition to genetic modification of the surface
protein to afford a reactive cysteine residue that was
labeled with fluorescent markers, the particle was
treated with an alkaline solution in order to remove
~20 coat protein subunits from the virus 5’-end,
exposing ~60 nucleotides. Such kind of particles
can be further annealed with complementary DNA
printed on a patterned microarray platform [64].
Using this strategy, Yi and co-workers prepared
encoded microparticles via nucleic acid hybridization
with mutant TMVs, which were suggested to have
broad applications in multiplexed target sensing [86].
Notably, all of the doubly-modified BNP scaffolds
consistently retained their structural integrity
throughout the reactions, which often involved a
mixture of aqueous and organic solvents. Further
studies of these BNPs are likely to involve in vitro
and in vivo models of cellular targeting, uptake and
drug delivery, but the anticipated immunogenic
response will be a major obstacle to overcome for
all protein-based scaffolds. To address this potential
problem, Manchester and co-workers employed
the CuAAC reaction to conjugate the folic acid-
PEG ligand to a mutant CPMV. While pegylation of
CPMV completely eliminated background binding
of the virus to tumor cells, the folate moiety allowed
CPMV-specific recognition of tumor cells bearing the
folate receptor [87].
2. Development of functional materials based on non-covalent interactions
The intrinsic chemistry of BNPs extends their
applications far beyond the basic covalent annealing
of different functional groups on the surface. Non-
covalent interactions (i.e., metal ligand, lectin
sugar, charge charge, π π stacking, and many other
interactions) have emerged as excellent approaches
for introducing novel functionalities to into BNPs.
BNP-based inorganic materials, greatly advanced
over the past years by Mann, Douglas, Young, and
Belcher, have made important contributions to the
synthesis of inorganic nanoparticles [32, 88 91].
Highly uniform, water-soluble protein shells provide
the added benefits of preventing aggregation and
sequestering mineralization reactions with various
metallic ions [92 94].
TMV, a rod-like BNP with a length of 300 nm
and 18 nm in diameter, which can be purified from
infected tobacco plants in large quantities, has been
exploited as template to grow metal or metal oxide
nanoparticles such as iron oxyhydroxides, CdS,
PbS, gold, nickel, cobalt, silver, copper, iron oxides,
CoPt, FePt 3 , and silica [95 102]. From electrophoretic
measurements, the isoelectric point of TMV is around
3.4 and so at neutral pH, the TMV surface has net
negative charge. In order to achieve successful
coating based on the electrostatic interactions, the
deposition conditions should be varied in order to
match the interaction between the virion surface
and the deposition precursor. In the case of silica
coating, carrying out the reaction at pH<3 results
in a positively charged TMV surface that will have
strong interactions with the anionic silicate sols
formed by hydrolysis of tetraethyl orthosilicate
(TEOS). In contrast, CdS, PbS, and iron oxides can
be successfully coated on the outer surface at near
neutral pH by specific metal ion binding with the
glutamate and aspartate residues [96]. As for metal
deposition, in some cases, a suitable activation agent
is needed in order to realize successful coating [101].
Pd(II) and Pt(II) are two typical activation agents.
The metal deposition can occur either inside the
inner channel or at the outer surface of TMV [101].
Genetically engineered TMV can show enhanced
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hydrolysis of Fe ( ) [91]. This strategy has also been
employed to encapsulate horseradish peroxidase
enzymes, where a single enzyme reactor was
constructed and the activities were studied at the
single-molecule level [116, 117].
To synthesize hybrid inorganic/viral core-shell
particles, it was demonstrated that inorganic particles
could serve as a core to nucleate the assembly of viral
capsids (Fig. 8(b)). For example, Dragnea and co-
workers used gold nanoparticles, quantum dots, and
magnetic nanoparticles to induce the organization of
the coat proteins of brome mosaic virus (BMV) [
121]. Therefore, nanoparticles can be encapsulated by
self-assembly in viral capsids, yielding VLPs in size
similar to the native virus. Furthermore, manipulation
of the diameter of the packaged material can be used
to vary the degree of packaging. Another strategy of
packaging nanoparticles in viral capsids was reported
by Franzen, where the origin of assembly sequences
was used as a trigger to package nanoparticles [122].
Association of these sequences on nanoparticles
provides recognition to the coat protein of wild-type
red clover necrotic mosaic virus (RCNMV), and self-
assembly of coat protein then begins until a VLP is
formed with the nanoparticles fully packaged within
the coat protein. The specific non-covalent interaction
between the RCNMV coat protein and the sequences
tethered on the nanoparticles offers great stability and
specificity in encapsidation of nanoparticles that can
be further explored for packaging other cargos [123].
In another study, virus-modified particles were
fabricated by the layer-by-layer approach and fused
with rubella virus-like particles (RVLPs) [124, 125].
Hence, it may be possible to fabricate particles
with virus functions at the surface of colloidal
particles along with other biological properties on
the nano/micro meter scale. It can be concluded
that non-covalent interactions, including layer-by-
layer techniques, may be a general approach for the
transfer of biological functionalities of various kinds
of viruses onto colloids, capsules, and flat surfaces
[126 129].
3. Discussion
The major features of BNPs that have been exploited
to generate novel biotemplates in the nanometer
range have been highlighted throughout the paper.
In short, their superb symmetry and size uniformity,
and system modularity in combination with genetic
engineering place BNPs as a material distinct from
polymeric materials. Viruses, ferritins, enzyme
complexes, chaperonines, and carboxysomes can
range widely in shape and size (spherical, rod-like,
from tens to hundreds of nanometers) with diverse
chemical and physical properties, thereby presenting
an extensive selection of primary building blocks.
Since these characterized BNPs only represent a
small fraction of the entire biodiversity, investigation
of other BNPs and their unique properties would be
of particular interest. For instance, viruses isolated
from extreme environments (e.g., high temperature)
would likely possess unparalleled thermal stabilities
which could allow for additional chemical reactions
at temperatures that would normally destroy other
BNP templates. Comparative analysis amongst other
BNPs may also elucidate which structural features
sequences would tolerate chemical and genetic
modifications, large peptide insertions, and structural
integrity.
Numerous studies have demonstrated the
potential of BNPs in biomedical applications made
possible by attaching ligands and small molecules to
redirect the protein carriers to different cell types for
drug/gene delivery and cell imaging. The nanosized
probes can further be modified with chemical
compounds, such as bio-imaging agents (near
infrared fluorescent dyes, magnetic contrast imaging
agents) and drugs at high local concentrations
to increase detection sensitivity and efficacy in
therapeutic applications. Furthermore, the BNPs can
be modified with polymers to boost their half-life in
the host, either by shielding the protein structures
from enzymatic degradation or the host’s immune
response.
Despite the advantages the BNPs possess, a
protein-based system exhibits similar limitations to
any other biological system. Unlike their inorganic
nanoparticle counterparts, the biological system
exhibits much lower stabilities at high temperatures,
in various organic solvents, and during long-term
storage. In addition, while these protein systems
possess the potential to generate libraries, only one
type of virus (bacteriophage M13) has a tested virus-
display and screening procedure. The possibility of
generating large arrays of mutant viruses, ferritins,
and BNPs and screening these systems against
specific targets is essential in order to realize the
full potential of these BNPs. Ultimately, through
the combination of these various functionalization
schemes, BNPs could be designed as versatile
templates with nanosized features for material
synthesis that would not be feasible via the
traditional top-down fabrication techniques.
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