Engineering Alkaline-Stable Barley Stripe Mosaic Virus-Like Particles for Efficient Surface Modification.

Viruses and virus-like particles are powerful templates for materials synthesis because of their capacity for precise protein engineering and diverse surface functionalization. We recently developed a recombinant bacterial expression system for the production of barley stripe mosaic virus-like particles (BSMV VLPs). However, the applicability of this biotemplate was limited by low stability in alkaline conditions and a lack of chemical handles for ligand attachment. Here, we identify and validate novel residues in the BSMV Caspar carboxylate clusters that mediate virion disassembly through repulsive interactions at high pH. Point mutations of these residues to create attractive interactions that increase rod length ~2 fold, with an average rod length of 91 nm under alkaline conditions. To enable diverse chemical surface functionalization, we also introduce reactive lysine residues at the C-terminus of BSMV coat protein, which is presented on the VLP surface. Chemical conjugation reactions with this lysine proceed more quickly under alkaline conditions. Thus, our alkaline-stable VLP mutants are more suitable for rapid surface functionalization of long nanorods. This work validates novel residues involved in BSMV VLP assembly and demonstrates the feasibility of chemical functionalization of BSMV VLPs for the first time, enabling novel biomedical and chemical applications.

Engineering Tobacco Mosaic Virus and Its Virus-Like-Particles for Synthesis of Biotemplated Nanomaterials.

Biomolecules are increasingly attractive templates for the synthesis of functional nanomaterials. Chief among them is the plant tobacco mosaic virus (TMV) due to its 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 and related viruses, 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. How these features have been exploited in engineering applications such as, sensing, catalysis, and energy storage are briefly outlined. While control of VLP surface features is well-established, fewer tools exist to control VLP self-assembly, which limits efforts to control template uniformity and synthesis of certain templated nanomaterials. However, emerging advances in synthetic biology, machine learning, and other fields promise to accelerate efforts to control template uniformity and nanomaterial synthesis enabling more widescale industrial use of VLP-based biotemplates.

BSMV as a Biotemplate for Palladium Nanomaterial Synthesis.

The vast unexplored virus biodiversity makes the application of virus templates to nanomaterial synthesis especially promising. Here, a new biotemplate, Barley stripe mosaic virus (BSMV) was successfully used to synthesize organic-metal nanorods of similarly high quality to those produced with Tobacco mosaic virus (TMV). The mineralization behavior was characterized in terms of the reduction and adsorption of precursor and nanocrystal formation processes. The BSMV surface-mediated reduction of Pd(2+) proceeded via first-order kinetics in both Pd(2+) and BSMV. The adsorption equilibrium relationship of PdCl3H2O- on the BSMV surface was described by a multistep Langmuir isotherm suggesting alternative adsorbate-adsorbent interactions when compared to those on TMV. It was deduced that the first local isotherm is governed by electrostatically driven adsorption, which is then followed by sorption driven by covalent affinity of metal precursor molecules for amino acid residues. Furthermore, the total adsorption capacity of palladium species on BSMV is more than double of that on TMV. Finally, study of the BSMV-Pd particles by combining USAXS and SAXS enabled the characterization of all length scales in the synthesized nanomaterials. Results confirm the presence of core-shell cylindrical particles with 1-2 nm grains. The nanorods were uniform and monodisperse, with controllable diameters and therefore, of similar quality to those synthesized with TMV. Overall, BSMV has been confirmed as a viable alternate biotemplate with unique biomineralization behavior. With these results, the biotemplate toolbox has been expanded for the synthesis of new materials and comparative study of biomineralization processes.

Decoupling and elucidation of surface-driven processes during inorganic mineralization on virus templates.

There is a lack of fundamental information about the molecular processes governing biomineralization of inorganic materials to produce nanostructures on biological templates. This information is essential for the directed synthesis of high quality nanomaterials via biotemplating. We characterized palladium (Pd) mineralization via the individual adsorption, reduction, and nanocrystal growth processes, which simultaneously occur during the hydrothermal synthesis on the Tobacco mosaic virus (TMV). The adsorption of precursor and reduction of palladium were decoupled through UV-vis Spectroscopy and in situ X-ray absorption spectroscopy studies. The role of additional cysteine (Cys) residues, ionic strength, and coating density on the fundamental parameters describing these processes were quantitatively evaluated. Primary nanocrystal growth and structural orientation of Pd nanoparticles was characterized using in situ small angle X-ray scattering. The adsorption, reduction of Pd species, and nanocrystal sizes were significantly changed on addition of Cys residues to the amino terminus of the TMV coat protein. Reduction of Pd on an already coated virion was dependent on the Pd surface area, and was hindered by the presence of residual salt. Furthermore, trends in Pd adsorption intensity and capacity suggested that chloride ions affected the adsorption equilibrium. Application of this fundamental approach with further optimization of parameters dictating biomineralization would facilitate directed synthesis and scale up of bioinorganic systems.

Mechanistic study of the hydrothermal reduction of palladium on the Tobacco mosaic virus.

The fundamental mechanisms governing reduction and growth of palladium on the genetically engineered Tobacco mosaic virus in the absence of an external reducer have been elucidated via in situ X-ray absorption spectroscopy. In recent years, many virus-inorganic materials have been synthesized as a means to produce high quality nanomaterials. However, the underlying mechanisms involved in virus coating have not been sufficiently studied to allow for directed synthesis. We combined XAS, via XANES and EXAFS analysis, with TEM to confirm an autocatalytic reduction mechanism mediated by the TMV1Cys surface. This reduction interestingly proceeds via two first order regimes which result in two linear growth regimes as spherical palladium nanoparticles are formed. By combining this result with particle growth data, it was discovered that the first regime describes growth of palladium nanoparticles on the virion while the second regime describes a second layer of larger particles which grew sporadically on the first palladium nanoparticle layer. Subsequent aggregation of free solution based spherical particles and metallized nanorods characterize a third and final regime. At the end of the second reduction regime, the average particle diameter of particles tethered to the TMV1Cys surface are approximately 4.5 nm. The use of XAS to simultaneously monitor the kinetics of biotemplated reactions along with growth of metal nanoparticles will provide insight into the pertinent reduction and growth mechanisms so that nanorod properties can be controlled through their populating nanoparticles.

The photosystem II oxygen-evolving complex protein PsbP interacts with the coat protein of Alfalfa mosaic virus and inhibits virus replication.

Alfalfa mosaic virus (AMV) coat protein (CP) is essential for many steps in virus replication from early infection to encapsidation. However, the identity and functional relevance of cellular factors that interact with CP remain unknown. In an unbiased yeast two-hybrid screen for CP-interacting Arabidopsis proteins, we identified several novel protein interactions that could potentially modulate AMV replication. In this report, we focus on one of the novel CP-binding partners, the Arabidopsis PsbP protein, which is a nuclear-encoded component of the oxygen-evolving complex of photosystem II. We validated the protein interaction in vitro with pull-down assays, in planta with bimolecular fluorescence complementation assays, and during virus infection by co-immunoprecipitations. CP interacted with the chloroplast-targeted PsbP in the cytosol and mutations that prevented the dimerization of CP abolished this interaction. Importantly, PsbP overexpression markedly reduced virus accumulation in infected leaves. Taken together, our findings demonstrate that AMV CP dimers interact with the chloroplast protein PsbP, suggesting a potential sequestration strategy that may preempt the generation of any PsbP-mediated antiviral state.

Phosphorylation of alfalfa mosaic virus movement protein in vivo.

The 32-kDa movement protein, P3, of alfalfa mosaic virus (AMV) is essential for cell-to-cell spread of the virus in plants. P3 shares many properties with other virus movement proteins (MPs); however, it is not known if P3 is posttranslationally modified by phosphorylation, which is important for the function of other MPs. When expressed in Nicotiana tabacum, P3 accumulated primarily in the cell walls of older leaves or in the cytosol of younger leaves. When expressed in Pischia pastoris, P3 accumulated primarily in a soluble form. Metabolic labeling indicated that a portion of P3 was phosphorylated in both tobacco and yeast, suggesting that phosphorylation regulates the function of this protein as it does for other virus MPs.

Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA.

To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

Arabidopsis thaliana is an asymptomatic host of Alfalfa mosaic virus.

The susceptibility of Arabidopsis thaliana ecotypes to infection by Alfalfa mosaic virus (AMV) was evaluated. Thirty-nine ecotypes supported both local and systemic infection, 26 ecotypes supported only local infection, and three ecotypes could not be infected. No obvious symptoms characteristic of virus infection developed on the susceptible ecotypes under standard conditions of culture. Parameters of AMV infection were characterized in ecotype Col-0, which supported systemic infection and accumulated higher levels of AMV than the symptomatic host Nicotiana tabacum. The formation of infectious AMV particles in infected Col-0 was confirmed by infectivity assays on a hypersensitive host and by electron microscopy of purified virions. Replication and transcription of AMV was confirmed by de novo synthesis of AMV subgenomic RNA in Col-0 protoplasts transfected with AMV RNA or plasmids harboring AMV cDNAs.