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\title{Engineering Tobacco Mosaic Virus, Barley Stripe Mosaic Virus, and their
Virus-Like-Particles for Synthesis of Biotemplated Nanomaterials}



\author[1]{Kok Zhi Lee}%
\author[2]{Vindula Basnayake Pussepitiya}%
\author[1]{Yu-Hsuan Lee}%
\author[1]{Sue Loesch-Fries}%
\author[1]{Michael Harris}%
\author[2]{Shohreh Hemmati}%
\author[1]{Kevin  Solomon}%
\affil[1]{Purdue University}%
\affil[2]{Oklahoma State University Stillwater}%


\vspace{-1em}



  
  \date{November 01, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Biomolecules are increasingly attractive templates for the synthesis of
functional nanomaterials. Chief among them are the plant Tobacco mosaic
virus (TMV) and Barley stripe mosaic virus (BSMV) due to their high
aspect ratio, narrow size distribution, diverse biochemical
functionalities presented on the surface, and compatibility with a
number of chemical conjugations. These properties are also easily
manipulated by genetic modification to enable the synthesis of a range
of metallic and non-metallic nanomaterials for diverse applications.
This article reviews the characteristics of TMV, BSMV, and their
virus-like particle (VLP) derivatives and how these may be manipulated
to extend their use and function. A focus of recent efforts has been on
greater understanding and control of the self-assembly processes that
drive biotemplate formation. We briefly outline how these features have
been exploited in engineering applications such as sensing, catalysis,
and energy storage, and discuss emerging advances that promise to
accelerate the development of these biotemplates for widescale
industrial use.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

Nanoscale materials offer precise tuning of material properties through
atomistic control of matter and energy interactions at increasingly
small length scales. This precision in material properties enables vast
new opportunities in sensing, data storage, energy storage, and
catalysis, among other areas {[}1--5{]}. Key to this process is the
synthesis of nanoscale materials with well-defined and uniform
architectures {[}4, 6{]}. Traditional chemical and physical synthesis
technologies rely on purely material- and energy-intensive processes
that are difficult to control and scale, rely on toxic or otherwise
non-green chemicals, have non-uniform outputs, and limited control of
atomistic features {[}7--10{]}. In contrast, biology synthesizes uniform
nanoscale biomolecules via well-defined design rules, which may be
engineered and serve as biotemplates for the synthesis of metallic
nanomaterials {[}11, 12{]}. Biomolecules, such as nucleic acids,
microtubules, amyloid fibers, and viruses, have been used as scaffolds
for the construction of hierarchical complex nanomaterials {[}13--17{]}.
Their surfaces present diverse biochemical functionalities that are used
to organize nanoparticle synthesis, and may be modified via conjugation
with organic or inorganic materials to create novel devices and control
metal mineralization {[}18, 19{]}. Finally, biomolecules possess
well-defined nanoscale architectures, are structurally stable across a
wide range of conditions, and can be easily manipulated via genetic
engineering. All these features make biomolecules attractive
biotemplates for bottom-up nanomaterial assembly.

Viruses possess many advantages over other types of biomolecules for
nanoparticle synthesis as they occur in a wide range of shapes and
sizes, and present diverse chemical functionalities for synthesis. Plant
viruses are widely used because they are harmless to human beings
{[}11{]}. For instance, Cowpea chlorotic mottle virus, Cowpea mosaic
virus, and Brome mosaic virus form icosahedral structures that range in
size from 18-30 nm while Tobacco mosaic virus and Barley stripe mosaic
virus assume rod-shaped structures up to 300 nm in length {[}11{]}. This
diversity enables biotemplating of diverse nanomaterials for
incorporation as catalysts, sensors, battery anodes, and semiconductor
digital memory devices {[}1--5{]}. Viral particles consist of
self-assembled capsid proteins (CPs) and nucleic acids that genetically
encode the CPs. The CPs present diverse biochemical functionalities via
amino acid residues on the particle surface that interact with metals in
solution and drive nanoparticle synthesis. These residues may be
conjugated to other compounds to enable synthesis of different
nanomaterials and create novel functional properties {[}18, 19{]}.
Similarly, the presented protein functionalities and dimensions can be
directly modified via engineering the encoding nucleic acid sequence
without dramatically altering viral structure to enable synthesis of new
materials {[}1, 20, 21{]}.

Non-infectious virus-like particles (VLPs) may be generated via
heterologous expression of CPs in non-native species without using the
complete viral genome {[}22{]}. Expressed CPs spontaneously
self-assemble into VLPs that possess the same rich chemical diversity on
their surfaces to drive nanoparticle synthesis. Plant VLPs also offer
several compelling features over real viruses for VLP engineering and
industrial-scale production. First, VLPs are more tolerant of mutations
than live virus enabling more engineering opportunities to enhance
function. For example, genetic modifications that enhance particle
structural stability to improve nanomaterial synthesis yields enable the
formation of nucleic acid-free VLPs that are unable to infect host cells
{[}23{]}. VLP production does not rely on infection for production and
may be stably produced with this enhancement in a heterologous host.
Second, heterologous microbial hosts replicate and produce CPs much more
rapidly than plants, which need several weeks to grow and mature before
infection with virus for production {[}24{]}. Moreover, live viruses are
infectious agents and must be grown in a Biosafety Level 2 greenhouse by
plant virologists to contain potential environmental contamination,
adding to their costs {[}25{]}. Third, bacterial VLP production also
leverages a wealth of bioprocessing infrastructure that has been
developed for large scale production of food, pharmaceuticals, and
chemicals {[}26{]}. Thus, VLPs are more compelling platforms for the
development of viral biotemplates.

Tobacco mosaic virus (TMV) is widely used for biotemplating due to its
architecture and physicochemical properties (Table 1). The dimensions of
TMV are well suited to biotemplating applications such as the production
of batteries and sensors {[}1, 6, 27{]}. TMV is self-assembled from over
a thousand copies of a single CP into a 300 nm long nanotube whose inner
and outer diameters are 4 nm and 18 nm, respectively {[}28{]}. This
aspect ratio maximizes the available surface area in compact volumes
enabling more efficient battery electrodes with higher charge densities,
and increased sensitivity to chemical analytes as sensors. Moreover, the
biochemical/physicochemical properties of TMV enable reduction of metal
ions and nanoparticle synthesis on the template under ambient conditions
{[}3, 29{]}. Finally, TMV and its VLPs consist of a single CP that is
amenable to genetic and chemical modifications that expand the types of
nanomaterials that may be synthesized, enhance morphological uniformity,
and increase particle density {[}30{]}. While TMV is the most commonly
used in bionanotechnology, the evolutionarily-related Barley stripe
mosaic virus (BSMV) provides a promising alternative biotemplate
{[}16{]}. BSMV has similar architecture to TMV (Table 1) but presents
distinct surface functional groups that accelerate nanoparticle
synthesis and increase nanoparticle density for increased electrical and
thermal conductivities and analyte sensitivity as sensors {[}16{]}.
Moreover, the evolutionary similarities between TMV and BSMV may allow
successful engineering strategies from TMV to be applied in BSMV to
expand and enhance the properties of BSMV-derived biotemplates. Thus,
BSMV is emerging as an attractive virus biotemplate for nanomaterial
synthesis.

The convergence of advances from materials science, structural biology,
molecular biology, chemistry, machine learning, engineering, and
synthetic biology, now enable the rapid engineering and development of
TMV, BSMV, and their VLPs for biotemplating. Their physicochemical
properties make them well-suited for the synthesis of diverse
nanomaterials for applications such as catalysis, energy storage, and
sensing. In this review, we provide an overview of the properties and
use of TMV, BSMV and their VLPs for nanoparticle synthesis, and focus on
emerging technologies and approaches to engineer VLPs to enhance their
function and broaden the nanomaterials that may be synthesized.

\textbf{2. Viral particle self-assembly and metal nanoparticle
synthesis}

Viral biotemplates such as TMV and BSMV spontaneously self-assemble from
identical CP subunits due to several covalent, hydrogen, and
electrostatic interactions encoded within the CP primary sequence and
its associated nucleic acids (Figure 1) {[}34{]}. These interactions
must be strong enough to withstand the pHs, temperatures, and ionic
strengths required for successful nanoparticle synthesis. CP is first
translated and folded before ultimately forming flat disks via
hydrophobic interactions between residues on the CP surface {[}34{]}. An
RNA sequence naturally found in the TMV viral genome, known as the
origin of assembly sequence or OAS, assumes a hairpin secondary
structure that then serves as a nucleus for nanotube formation. CP disks
are threaded by OAS-containing RNA, allowing for electrostatic
interactions between the OAS and CP {[}35{]}. This interaction forces
the CP disk to assume a helical conformation, reorienting themselves by
induced proton adsorption and subsequent hydrogen-bonding, which then
rapidly polymerizes with other CP molecules into a nanotube {[}34{]}.
Strengthening the final assembly are electrostatic interactions between
adjacent CPs, which are mediated by a handful of negatively charged
residues in a motif known as the Caspar carboxylate cluster {[}36{]}.
Calcium ions typically neutralize the repulsive negative charges of
adjacent CPs and drive viral polymerization. Control of these
interactions via genetic engineering of the CP and/or the OAS offers
tremendous potential to modify nanotube architecture and its stability
in subsequent biotemplating {[}23, 37{]}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Driving forces for TMV assembly. TMV CP experiences
various interactions, including hydrophobic interactions, RNA
initiation, electrostatic interactions in the Caspar carboxylate cluster
(CCC), hydrogen bonding, and RNA-protein interaction at different stages
of assembly.

Metal nanoparticles are frequently synthesized spontaneously on viral
biotemplates using aqueous metal solutions. The metal precursor ions
adsorb and are chemically reduced on the viral particle surfaces at many
adsorption and nucleation sites to form a metallic nanomaterial {[}38,
39{]}. The chemical interactions that drive metal precursor adsorption
and reduction are not well understood. However, the adsorption process
is frequently described by a single-step Langmuir isotherm that is
solely driven by covalent interactions, e.g. palladium on TMV {[}38{]}.
As metal ion precursor adsorption and reduction are the fundamental
processes that drives metal coating formation, the oxidation potential
of surface accessible residues must be sufficient to drive metal
reduction (Table 2). Amino acid residues that are easily oxidized, such
as cysteine, tyrosine, and lysine, more readily interact with metals
driving deposition {[}40--42{]}. Metals with higher positive reduction
potentials, including gold, silver, and platinum, can be reduced by the
various functional groups present on the CP of TMV (Table 2) {[}39{]}.
This deposition is frequently enhanced by engineering the amino acids
residues that are presented on the virus/VLP {[}30, 39{]}. Other metals
such as nickel, iron, and cobalt cannot be reduced this way as they have
negative reduction potentials. Instead, a different metal that is more
readily reduced such as palladium is mineralized first onto the CP,
which then serves as a catalyst to reduce target metal ions to metal
atoms {[}43, 44{]}. For example, nickel and cobalt are deposited in the
inner channel of TMV after mineralization of TMV with Pd and Pt
{[}44{]}. Fundamentally, appropriate pairing of amino acid side chain
that can chemically reduce and interact with metals intrinsically sets
the metal adsorption capacity and controls the rate of reaction as
nanoparticle synthesis proceeds spontaneously under ambient conditions.

Plant-produced BSMV has been demonstrated to be a viable biotemplate for
mineralization of palladium nanowires, however, biomineralization with
BSMV differs from that of TMV {[}16{]}. The surface of BSMV allows metal
ion precursor deposition to proceed via a multi-step Langmuir isotherm
that incorporates both electrostatic and covalent adsorbent-adsorbate
interactions. This difference may arise in part due to the larger amount
of BSMV surface-exposed residues, compared to TMV, in an unstructured
insertion loop containing 10 amino acids that protrudes from the
particle surface {[}33{]}. These stronger interactions increase the
adsorption capacity for Pd on BSMV two-fold compared to that on TMV
{[}16{]}. Similarly, the rate of adsorption is increased compared to
TMV, suggesting that BSMV can be fully coated in fewer processing cycles
saving both time and expensive precursor material. Furthermore, BSMV
biotemplates produce more uniformly sized nanoparticles relative to TMV.
The additional opportunities to engineer metal deposition via the
insertion loop and superior adsorption and metal nanoparticle synthesis
characteristics make BSMV an attractive alternative to TMV that may
generate more uniform metal nanostructures more economically.

\textbf{3. Nanostructure Synthesis with Viral Biotemplates for Diverse
Applications}

Nanostructures are increasingly sought to create advanced materials that
improve electrical conductivity and capacitance, current generation,
catalytic activity, and detection sensitivity {[}3, 5, 19, 46, 47{]}.
These applications benefit from high aspect ratio nanomaterials that can
be biotemplated on TMV, BSMV or their VLPs. However, each application
relies on distinct metals whose unique electronic structure is key to
enabling these applications; e.g. metal with high conductivity such as
copper is used in conductive nanowires while gold is relatively inert
making it an ideal substrate for a sensor {[}19, 48{]}. Moreover, the
precise function of the nanomaterial in these roles relies on their
incorporation into devices with unique architectures (Figure 2). To
enable the synthesis of these nanomaterials, the viral biotemplates are
modified and processed in innovative ways; we describe only a handful
below.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2} . Some potential applications of nanomaterials
templated on TMV, BSMV and their VLPs.

\textbf{3.1 Metal Nanowires}

Metal nanowires are used for a multitude of applications from
electronics and sensors to lasers due to their improved conductivity
{[}49{]}. Industrial-scale production of metal nanowires is most
commonly achieved via polyol synthesis. However, it is not
environmentally-friendly or sustainable as it uses various toxic
polyhydric reducing agents such as ethylene glycol and propylene glycol
in an energy-intensive reaction process that occurs at high temperatures
{[}8, 50--52{]}. In contrast, highly conductive and uniform nanowires
may be synthesized without toxic reagents or high temperatures via
templating on TMV or its VLP {[}53{]}. To enable creation of palladium,
platinum or gold nanowires, additional lysine, cysteine, and/or tyrosine
side chains are added to the surface of the viral template to provide
sufficient reducing power for nanoparticle synthesis and potential
nucleation sites for metal binding. The introduction of this reduction
power can decrease or completely remove the need for additional reducing
agents in nanowire synthesis, thereby lowering costs {[}29, 54{]}.

For thinner nanowires, metal may be deposited in the narrow viral
channel of TMV, rather than on the surface {[}55, 56{]}. Buffering
agents are used to alter the ionic state of amino acid side chains,
changing its electrostatic interactions to decrease metal deposition on
the surface. The inner channel, whose side chains are distinct from the
surface, remain charged and electrostatically interact with metal
solutions to adsorb and reduce synthesized metal nanoparticles. These
techniques may be directly applied to platinum or palladium nanowires;
however, nanowires composed of other metals such as copper, cobalt,
and/or nickel require a core of Pd and Pt, which then serves as
catalytic sites for reduction of other metals forming thin alloy
nanowires such as CoFe, CoNi, FeNi, and CoFeNi {[}44, 55{]}. Bimetallic
ferromagnetic alloys with Pt such as CoPt and FePt\textsubscript{3} may
also be synthesized directly by mixing precursor metal salt solutions
with platinum or palladium salts in the same deposition bath thus
simplifying the two-step process {[}43{]}.

\textbf{3.2 Batteries and Energy Storage}

Batteries produce current via chemical reactions on the surface of
electrodes, which is limited by the available surface area {[}3{]}. To
generate the currents needed to power modern electronic devices, metal
coated TMV virions can be vertically oriented on top of a gold substrate
to form a carpet-like nanoforest structure (Figure 2). The vertical
attachment is achieved by introducing a cysteine residue at the amino
terminus of CP to produce TMV1cys virions {[}3, 6{]}. Most cysteines in
TMV are partially recessed and thus are incapable of metal binding;
however, the N-terminal residue is fully exposed allowing for
near-vertical assembly of the biotemplate on a gold substrate via
covalent interactions between the gold and the thiol group of the
cysteine. Subsequent coating with nickel and/or cobalt increases the
active electrode surface area more than ten-fold with a doubling in
electrode discharge capacity (or current generation) {[}3{]}. Similarly,
Pd/Ni/Si-coated TMV anodes in lithium ion batteries can increase the
discharge capacity by nearly ten-fold compared to then available
graphite anodes {[}6{]}. Finally, incorporating TMV in sodium-ion
batteries as a carbon/tin/nickel-coated TMV anode can increase battery
cycling lifespan with little degradation in charge capacity over 150
deep charging cycles. This capability made it the longest-cycling
nano-Sn anode material for Na-ion batteries at the time {[}27{]}. Coated
TMV patterned with a similar nanoforest structure has also been shown to
increase the performance of micro supercapacitors (Figure 2), which also
rely on surface area to store charges {[}57{]}.

\textbf{3.3 Catalysis}

Catalyst effectiveness is proportional to its exposed surface area, as
more sites are available for reaction. To minimize the costs of large
volumes of catalytic metal, an economical strategy for catalyst
synthesis is to deposit catalytic metal on an inexpensive inert
substrate with a high surface area to volume ratio such as carbon or a
biotemplate. However, biotemplated catalysts are more efficient; Pd
catalyst synthesized from TMV1cys immobilized on a gold substrate in a
nanostar configuration increased the reaction rate 68\% relative to
commercial Pd/C catalyst of comparable size {[}58{]}. TMV-templated
catalysts are stable and can be recycled with negligible degradation in
performance over several cycles {[}4, 5, 58{]}. The superior performance
of TMV-templated catalyst is attributed primarily to two factors. One,
palladium particles mineralized on TMV tend to be more uniform as
compared to commercially available palladium catalyst supported on
carbon material leading to more efficient catalysis {[}58, 59{]}. And
secondly, fabrication of palladium on TMV does not use surfactants and
capping agents like the synthesis of traditional catalysts that can
block active sites {[}60{]}. Removal of these surfactants to rescue
catalysis is incomplete and adds to the cost of catalyst manufacture.
Thus, biotemplated catalysts are both more catalytically- and
economically- advantaged in many scenarios.

\textbf{3.4 Sensors}

Sensors are used in a wide range of areas including environmental and
disease monitoring to detect and quantify selected analytes from complex
dirty solutions. Effective sensors must have high selectivity,
sensitivity, and structural stability over the range of environmental
conditions they are likely to experience. Selectivity for a specific
analyte in mixed samples is achieved by functionalizing the viral
surface with metals, enzymes and/or other chemicals. For example,
palladium-coated TMV rapidly and reversibly detects hydrogen at room
temperatures {[}47{]}. Similarly, gold coated nanowires conjugated with
folic acid via coupling agents can interact with the folate receptors on
tumor cells to detect cancers with high sensitivity {[}19{]}.
Additionally, TMV may be decorated with biotin to enable binding of
enzymes to detect penicillin {[}61{]}. Other desirable sensor properties
such as sensitivity and stability arise from the nature of viral
biotemplates themselves. TMV's high surface area to volume ratio allows
for more contact with analytes, amplifying signal generation and
improving device sensitivity {[}62{]}. Similarly, TMV structure is very
stable in high temperatures and over a wide range of pHs (Table 1) that
may be encountered in the analysis of complex mixtures, making
biosensors derived from TMV more durable and rugged.

\textbf{4. Engineering Viruses and VLPs for Enhanced Biotemplate
Properties}

Improved understanding of TMV and BSMV structural biology and molecular
biology has informed a number of strategies to control and enhance
biotemplate properties. These include improved structural stability to
enable synthesis of more diverse nanomaterials, control of VLP
architecture and enhanced uniform coating to control physical
properties, and more versatile conjugation capabilities to tune
function. These modifications are powered by the flexibility of the
CP-encoding nucleic acids, which can be readily engineered via genetic
tools.

\textbf{4.1 Structural stability in high temperatures and extreme pH}

Despite the successes in coating TMV, BSMV and their VLPs with metals
such as gold, palladium and platinum, many industrially-desirable metals
are not readily deposited due to unfavorable electrochemistries (Table
2) {[}30, 63{]}. This issue may be mitigated somewhat through the use of
buffering and reducing agents, activation by primary deposition with
another metal such as Pt, and higher temperatures to create more
favorable processing conditions. These strategies ultimately increase
metal-template interactions via altering residue ionization state,
changing the reduction potentials of the metal ions and amino acid side
chains, and/or create nucleation sites to drive metal mineralization.
However, these conditions may destabilize the CP interactions that drive
self-assembly of the template leading to biotemplate loss and low yields
of metal mineralization. Biotemplates are only stable within fixed pH
and temperature ranges (Table 1). To address this challenge, TMV and
VLPs have been engineered to increase their structural stability to
resist disassembly.

The stability of TMV biotemplates has been increased by control of the
intermolecular forces that drive self-assembly (Figure 1), enabling more
rapid biotemplating of a wider range of materials {[}64--66{]}. For
example, single point mutations within the Caspar carboxylate cluster
can enhance viral assembly. Neutralization of a negatively-charged
residue (E50Q) or replacing a negatively-charged residue with a
positively-charged residue in the Caspar carboxylate center (E50R or
D77R) has been shown to produce longer virions that spontaneously
self-assemble without an RNA that contain an OAS or other nucleic acids
in transgenic plants {[}67{]}. In similar fashion, neutralizing the
negative-charged residues with site-specific mutations, E50Q/D77N, was
sufficient to rescue TMV-VLP assembly in \emph{E.coli} even in the
absence of RNA with an OAS, which typically results in viral disks alone
{[}21{]}. In combination with cysteine engineering, E50Q/D77N mutants
are able to attach to a gold-coated plate {[}21{]}. These mutations
within the Caspar carboxylate cluster are sufficient to overcome the
poor stability of nucleic acid-free VLPs and recover resistance to pH,
attaining similar stability to wildtype virions. In so doing, they
create nucleic acid-free VLPs that have a free internal channel for
synthesis of thin nanowires {[}64{]}. Engineering of the hydrophobic
interactions between CPs (Figure 1) may further improve the stability of
VLPs; however, modifications of this nature have yet to be evaluated.

OAS-containing nucleic acids that initiate the assembly of wildtype
virus and VLPs also act as a molecular ruler that sets the length of
produced VLPs {[}20{]}. While VLPs that have been engineered
appropriately (e.g. at the Caspar carboxylate cluster) can self-assemble
without this molecular ruler, the resulting VLPs are more heterogenous
in size than OAS-containing viral particles with sizes as small as 20 nm
{[}23{]}. Thus, future work should include efforts to control the
precise dimensions of VLPs or development of efficient separation
technologies to isolate VLPs with specific dimensions.

\textbf{4.2 Coating uniformity}

Nanomaterial properties are controlled in part by the coating uniformity
or smoothness of the surface of metal deposited on biotemplate. This
uniformity relies on the molecular interactions between the deposited
metal and residues on the surface or in the channel of the biotemplate.
Coating agents such as citric acid are added to the deposition reaction
to alter the charge of surface-exposed residues and increase
metal-biotemplate interactions during the metal deposition process
{[}39, 68, 69{]}. However, these pH-sensitive electrostatic interactions
can be disrupted by other reagents of the deposition process leading to
poor nanoparticle synthesis and nonuniform coating.

To address this challenge, covalent bonds that are resistant to modest
pH changes can be introduced to drive nanoparticle synthesis. Site
specific mutagenesis of the capsid protein to introduce cysteine and
lysine residues has been demonstrated to improve nanoparticle synthesis
{[}30, 70{]}. These residues may be introduced either on the surface or
inner channel of TMV VLPs (e.g. S3C, T103C, T158K) or on either terminus
of the CP {[}70--72{]}. These modifications allow for metal deposition
under a wider range of pHs and promote high density nucleation sites for
nanoparticle synthesis, unlike wildtype whose nanoparticle synthesis is
driven by weaker surface drying effects. For example, two cysteine
residues have been genetically added to the amino-terminus of TMV CP,
resulting in 4260 available thiol groups per VLP for metal reduction and
deposition {[}30{]}. The introduced thiol groups on the TMV surface
serve as a reaction site for nanomaterials such as gold and palladium to
reduce metal ions or form covalent bonds with non-metal materials via
thiol coupling. Cysteine enhances the deposition of several metals
including gold, silver, and palladium, and also enables novel
attachments for device manufacture (e.g. nanoforests for battery anode
synthesis) {[}30{]}. Lysine substitution has also served as a functional
group for chemical conjugation of silica coatings {[}70{]}.

\textbf{4.3 Diversifying the nanomaterials that can be synthesized on
TMV/BSMV-derived biotemplates}

While TMV/BSMV virions and VLPs serve as exceptional biotemplates for
metal mineralization, they are not directly compatible for the synthesis
of non-metallic nanomaterials. However, the introduction of
surface-accessible amino acids, including aspartate, glutamate, and
tyrosine provide conjugation sites for synthesis of non-metallic
nanomaterials via covalent bonding. For example, the carboxylic acid
moiety of aspartate and glutamate can react with amines, which have been
functionalized with biotin, chromophores, and crown ethers while
tyrosine can conjugate with PEG {[}73{]}. These conjugations enable
medical applications of TMV such as vectors for drug delivery.

In addition to canonical amino acids, synthetic noncanonical amino acids
with unique functional groups have also been incorporated in other VLPs
such as hepatitis B virus (HBV) or bacteriophage Q\selectlanguage{greek}β \selectlanguage{english}to expand coating
capabilities further {[}74{]}. Noncanonical amino acids provide unique
functional groups, such as alkynes and aminophenylalanine, that
facilitate bioorthogonal coating {[}75{]}. Bioorthogonal reactions
enable specific conjugation via the noncanonical amino acids without
cross-reaction with canonical amino acids of the virions/VLPs, reducing
the risk of disrupting the physicochemical properties that drive
biotemplate self-assembly. Alkyne-containing noncanonical amino acids
have been incorporated into virions to attach PEG, oligonucleotides,
antibody fragments, and other peptides via click chemistry {[}74{]}
while aminophenylalanine incorporated VLPs have been used to conjugate
cell type-specific targeting peptides, enabling drug delivery to
specific cell types {[}76{]}. The number of novel viral-derived
biomaterials are certain to expand further as new noncanonical amino
acids are engineered and incorporated into CPs {[}77{]}.

\textbf{5. Future directions}

Current advances in a number of fields now converge to enhance the
function and potential of viral biotemplates. These activities are
greatly accelerated by the innovations of synthetic biology that enable
more rapid building and testing of engineered VLPs. These constructs are
thoroughly characterized with spectroscopic techniques with the rich
datasets fed to machine learning algorithms to guide iterative design
and improvements (Figure 3). Several emerging opportunities are as
follows.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3.} Emerging opportunities to accelerate development of
TMV, BSMV and their VLPs. In the ``design'' phase, VLPs with specific
properties (e.g. conjugation to an enzyme via noncanonical amino acids)
for an application guide the design of DNA sequences that are then
rapidly assembled in the ``build'' phase. Advanced DNA synthesis methods
rapidly assemble DNA to produce thousands of variants while host
engineering boosts VLP production. In the ``test'' phase, these
constructs are expressed and screened. Directed evolution develops VLPs
with desirable features while cell-free systems enable high-throughput
screening. In the ``learn'' phase, characterization of the resulting
products and analysis via empirical and machine learning approaches
refine the initial design ultimately leading to an optimal biotemplate
and bioproduction platform.

\textbf{5.1 Rapid prototyping and screening of novel biotemplates}

Despite the exciting future for VLP development, the production and
characterization of newly engineered VLPs are time-consuming processes,
spanning several days. Thus, new screening tools are necessary to
expedite the development of newly engineered VLPs. Cell-free systems
that contain the transcriptional and translational machinery needed for
VLP production could become integral to rapid VLP prototyping and
characterization without laborious purification. Cell-free systems can
be used to produce engineered VLP candidates in a highly parallel or
high-throughput manner with minimal inputs due to their microliter scale
reaction volumes {[}78{]}. VLPs derived from non-plant viruses are
already being developed via cell-free technologies. For example, a
cell-free system was used to incorporate noncanonical amino acids in
bacteriophage MS2 and bacteriophage Q\selectlanguage{greek}β \selectlanguage{english}for click-chemistry
functionalization {[}79{]}.

Rapid screening is also needed to identify engineered mutants with
desirable properties. Advanced DNA synthesis methods such as Gibson and
Golden-gate assemblies can produce thousands of DNA-encoding VLPs
variants in a single reaction {[}80--82{]}. However, screening of these
variants for improved function requires characterization of individual
mutants, which makes the screening process slow and inefficient. Instead
of screening every variant, directed evolution can be applied to evolve
mutants with desirable traits {[}83{]}. Directed evolution is a process
where mutants are propagated and those with desirable properties are
selected for. The ability of a given mutant to propagate or replicate is
linked to the property that is desired, allowing surviving mutants to
`select' for enhanced properties. Thus, thousands of variants can be
simultaneously evaluated in hours without screening of every single
variant. The selected variants can then be mutated to introduce
additional genetic diversity and subject to another round of selection.
The iteration of these processes can generate protein mutants with
optimized or even new properties such as producing VLPs with enhanced
structural stability {[}84{]}. Although directed evolution has not been
widely used for VLP engineering, the recent development of new directed
evolution methods, including assisted machine learning {[}85{]}, which
identifies more promising mutations to be constructed and screened,
and\emph{in vivo} continuous directed evolution {[}86{]}, which
streamlines the genetic engineering process, will accelerate the
engineering of VLPs.

\textbf{5.2 Improved biotemplate production yields}

VLPs frequently form insoluble aggregates when expressed in non-native
hosts that are unsuitable for biotemplating processes {[}87{]}. To
increase the yield of usable VLP biotemplates, the solubility of
heterologous CP must be enhanced. There are many ways to increase
protein solubility {[}88{]}; however, increasing CP solubility for VLPs
production is challenging as one of the drivers of protein insolubility,
strong hydrophobic interactions, is also the driving force for virion
and VLP assembly. However, machine learning algorithms have begun to
identify the design rules for soluble VLP CP design without disrupting
VLP self-assembly based on hydrophobicity scales {[}89{]}. An early
report with these tools counterintuitively suggests that CP solubility
may be highly correlated with arginine content in some VLPs, despite its
negative correlation with solubility of short chain variable fragments.
Arginine reduces many hydrophobic protein-protein interactions while
interacting favorably with other amino acids to reduce random
aggregation. However, these tools have yet to be applied to forward
engineer novel VLP CPs.

\textbf{5.3 Molecular understanding and control of the mineralization
process}

While the basic processes of metal mineralization are qualitatively
understood, mechanistic details regarding the reaction mechanisms of
metal reduction and adsorption remain elusive. Current studies on metal
mineralization using TMV, BSMV and their corresponding VLPs as
biotemplates focused on characterization of synthesized nanostructures
using different techniques such as transmission electron microscopy
(TEM) {[}24{]}, Fourier transform infrared spectroscopy (FTIR), and
X-ray scattering analysis {[}19, 59{]}. These methods reveal the
structure of the final synthesized structure and inform hypotheses of
how metal mineralization has occurred. However, a more effective
approach to understand how metal mineralization takes place is to
perform \emph{in-situ} FTIR {[}90{]}. This allows direct observation of
the reaction progress enabling determination of the mechanism of
mineralization. It is also possible to observe how changes in reaction
conditions such as pH, temperature, and concentrations of precursors,
reducing agents and biotemplate would affect metal mineralization
including particle size, particle size homogeneity, and the type of
metal nanostructures mineralized on the surface. This would require an
extensive design of experiments to systematically evaluate the effect of
each parameter and their interactions. Machine learning algorithms such
as artificial neural networks have already been applied to these rich
datasets to create predictive models for nanoparticle synthesis as a
function of processing parameters {[}91{]}. Similarly, neural networks
first used to predict the binding of metallic ion cofactors to enzymes
could be extended to VLPs to predict and model metal-biotemplate
interactions as a function of engineered CP protein sequence {[}92{]}.
Such computational tools would greatly accelerate biotemplate
engineering efforts and optimize deposition processes for metallic
nanomaterial synthesis.

\textbf{6. Concluding remarks}

Integrated concepts from material science, biology, chemistry, and
engineering have developed TMV, BSMV, and their VLPs for the synthesis
of high-quality nanomaterials for diverse applications. TMV and BSMV are
particularly attractive as biotemplates due to their ease of
manipulation, large scale production in bacterial culture, and surface
moieties that facilitate interactions with a number of materials. New
insights afforded by structural biology and spectroscopic
characterization now inform the engineering of CP molecular interactions
within a viral particle/VLP, and between the viral particle/VLP and
deposited nanomaterials. These have led to a number of innovative
genetic strategies that enhance biotemplate structural stability and
improve the uniformity and properties of synthesized nanoscale
structures enabling diverse applications such as sensing, catalysis, and
energy storage. While further engineering is needed to scale up these
systems for widespread industrial nanomaterial synthesis, emerging
technologies from synthetic biology and machine learning promise to
accelerate the pace of TMV- and BSMV- derived biotemplate development in
the years to come.

\textbf{Acknowledgement}

This review was partially supported by the USDA National Institute of
Food and Agriculture (Hatch Multistate Project S1075) and the Robert B
and Virginia V. Covalt Endowed Professorship of Chemical Engineering at
Purdue. KZL is partially supported by Purdue Research Foundation
Fellowships (\#60000025 \& \#60000029) and a Ralph W. and Grace M.
Showalter Research Trust Award (\#41000622) to KVS. VBP is supported by
startup funds from the College of Engineering, Architecture and
Technology at Oklahoma State University. YHL is supported by the Robert
B and Virginia V. Covalt Endowed Professorship of Chemical Engineering
awarded to MTH. The funding sources had no role in the study design,
data collection, data analysis, decision to publish, or preparation of
the manuscript.

\textbf{Conflict of interest}

The authors declare no financial or commercial conflict of interest.

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\emph{}

\emph{Table 1. Characteristics of TMV and BSMV viral particles}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
& TMV & BSMV\tabularnewline
\midrule
\endhead
Outer diameter (nm) & 18\textsuperscript{a} &
20\textsuperscript{c}\tabularnewline
Inner diameter (nm) & 4\textsuperscript{a} &
3-4\textsuperscript{d}\tabularnewline
Length (nm) & 300\textsuperscript{a} &
110-150\textsuperscript{c}\tabularnewline
Thermal stability* () & 90\textsuperscript{a} &
70\textsuperscript{c}\tabularnewline
pH stability & 3.0-9.0\textsuperscript{a} & --\tabularnewline
CP size (kDa) & 17.6\textsuperscript{a} &
22\textsuperscript{c}\tabularnewline
Copies Of CP & 2130\textsuperscript{a} &
\textasciitilde{}1385\textsuperscript{+}\tabularnewline
isoelectric point (pH) & 3.9\textsuperscript{b} &
4.5\textsuperscript{c}\tabularnewline
\bottomrule
\end{longtable}

*Limit at which virus is no longer infectious.

\textsuperscript{a}{[}28{]};
\textsuperscript{b}{[}31{]};\textsuperscript{c}{[}32{]};
\textsuperscript{d}{[}33{]};\textsuperscript{+}estimated from helix
pitch and CPs per turn {[}25{]}

-- not measured

\emph{Table 2. Reduction potential of some metals at 25
C}{[}45{]}\emph{.}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Species & Half-Reaction & Reduction Potential (V)\tabularnewline
\midrule
\endhead
\textbf{Au} & Au\textsuperscript{3+} + 3e\textsuperscript{-} =
Au\textsubscript{(s)} & 1.52\tabularnewline
\textbf{Pt} & Pt\textsuperscript{2}+ + 2e\textsuperscript{-} =
Pt\textsubscript{(s)} & 1.20\tabularnewline
\textbf{Pd} & Pd\textsuperscript{2+} + 2e\textsuperscript{-} =
Pd\textsubscript{(s)} & 0.92\tabularnewline
\textbf{Ag} & Ag\textsuperscript{+} + e\textsuperscript{-} =
Ag\textsubscript{(s)} & 0.80\tabularnewline
\textbf{Cu} & Cu\textsuperscript{+} + e\textsuperscript{-} =
Cu\textsubscript{(s)} & 0.52\tabularnewline
\textbf{Ni} & Ni\textsuperscript{2+} + 2e\textsuperscript{-} =
Ni\textsubscript{(s)} & - 0.24\tabularnewline
\textbf{Fe} & Fe\textsuperscript{2+} + 2e\textsuperscript{-} =
Fe\textsubscript{(s)} & - 0.44\tabularnewline
\textbf{Co} & Co\textsuperscript{2+} + 2e\textsuperscript{-} =
Co\textsubscript{(s)} & - 0.28\tabularnewline
\textbf{Fe} & Fe\textsuperscript{3+} + 3e\textsuperscript{-} =
Fe\textsubscript{(s)} & - 0.04\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure Legends}

\textbf{Figure 1.} Driving forces for TMV assembly. TMV CP experiences
various interactions, including hydrophobic interactions, RNA
initiation, electrostatic interactions in the Caspar carboxylate cluster
(CCC), hydrogen bonding, and RNA-protein interaction at different stages
of assembly.

\textbf{Figure 2} . Some potential applications of nanomaterials
templated on TMV, BSMV and their VLPs.

\textbf{Figure 3.} Emerging opportunities to accelerate development of
TMV, BSMV and their VLPs. In the ``design'' phase, VLPs with specific
properties (e.g. conjugation to an enzyme via noncanonical amino acids)
for an application guide the design of DNA sequences that are then
rapidly assembled in the ``build'' phase. Advanced DNA synthesis methods
rapidly assemble DNA to produce thousands of variants while host
engineering boosts VLP production. In the ``test'' phase, these
constructs are expressed and screened. Directed evolution develops VLPs
with desirable features while cell-free systems enable high-throughput
screening. In the ``learn'' phase, characterization of the resulting
products and analysis via empirical and machine learning approaches
refine the initial design ultimately leading to an optimal biotemplate
and bioproduction platform.

\selectlanguage{english}
\FloatBarrier
\end{document}

