Ultrafast Collective Excited-State Dynamics of a Virus-Supported Fluorophore Antenna.

Radiation brightening was recently observed in a multifluorophore-conjugated brome mosaic virus (BMV) particle at room temperature under pulsed excitation. On the basis of its nonlinear dependence on the number of chromophores, the origins of the phenomenon were attributed to a collective relaxation. However, the mechanism remains unknown.

Subset of Fluorophores Is Responsible for Radiation Brightening in Viromimetic Particles.

In certain conditions, dye-conjugated icosahedral virus shells exhibit suppression of concentration quenching. The recently observed radiation brightening at high fluorophore densities has been attributed to coherent emission, , to a cooperative process occurring within a subset of the virus-supported fluorophores. Until now, the distribution of fluorophores among potential conjugation sites and the nature of the active subset remained unknown.

Virus Assembly Pathways: Straying Away but Not Too Far.

Non-enveloped RNA viruses pervade all domains of life. In a cell, they co-assemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged.

Disassembly Intermediates of the Brome Mosaic Virus Identified by Charge Detection Mass Spectrometry.

Capsid disassembly and genome release are critical steps in the lifecycle of a virus. However, their mechanisms are poorly understood, both in vivo and in vitro. Here, we have identified two in vitro disassembly pathways of the brome mosaic virus (BMV) by charge detection mass spectrometry and transmission electron microscopy.

Radiation Brightening from Virus-like Particles.

Concentration quenching is a well-known challenge in many fluorescence imaging applications. Here, we show that the optical emission from hundreds of chromophores confined onto the surface of a 28 nm diameter virus particle can be recovered under pulsed irradiation. We have found that as one increases the number of chromophores tightly bound to the virus surface, fluorescence quenching ensues at first, but when the number of chromophores per particle is nearing the maximum number of surface sites allowable, a sudden brightening of the emitted light and a shortening of the excited-state lifetime are observed.

In Vitro Assembly of Virus-Derived Designer Shells Around Inorganic Nanoparticles.

Nanoparticle-templated assembly of virus shells provides a promising approach to the production of hybrid nanomaterials and a potential avenue toward new mechanistic insights in virus phenomena originating in many-body effects, which cannot be understood from examining the properties of molecular subunits alone. This approach complements the successful molecular biology perspective traditionally used in virology, and promises a deeper understanding of viruses and virus-like particles through an expanded methodological toolbox. Here we present protocols for forming a virus coat protein shell around functionalized inorganic nanoparticles.

Defects and Chirality in the Nanoparticle-Directed Assembly of Spherocylindrical Shells of Virus Coat Proteins.

Virus coat proteins of small isometric plant viruses readily assemble into symmetric, icosahedral cages encapsulating noncognate cargo, provided the cargo meets a minimal set of chemical and physical requirements. While this capability has been intensely explored for certain virus-enabled nanotechnologies, additional applications require lower symmetry than that of an icosahedron. Here, we show that the coat proteins of an icosahedral virus can efficiently assemble around metal nanorods into spherocylindrical closed shells with hexagonally close-packed bodies and icosahedral caps.

Toward Virus-Like Surface Plasmon Strain Sensors.

The strong configuration dependence of collective surface plasmon resonances in an array of metal nanoparticles provides an opportunity to develop a bioinspired tool for sensing mechanical deformations in soft matter at the nanoscale. We study the feasibility of a strain sensor based on an icosahedral array of nanoparticles encapsulated by a virus capsid. When the system undergoes deformation, the optical scattering cross-section spectra as well as the induced electric field profile change.

Examining the Heterogeneous Genome Content of Multipartite Viruses BMV and CCMV by Native Mass Spectrometry.

Since the concept was first introduced by Brian Chait and co-workers in 1991, mass spectrometry of proteins and protein complexes under non-denaturing conditions (native MS) has strongly developed, through parallel advances in instrumentation, sample preparation, and data analysis tools. However, the success rate of native MS analysis, particularly in heterogeneous mega-Dalton (MDa) protein complexes, still strongly depends on careful instrument modification. Here, we further explore these boundaries in native mass spectrometry, analyzing two related endogenous multipartite viruses: the Brome Mosaic Virus (BMV) and the Cowpea Chlorotic Mottle Virus (CCMV).

Measurement of Nanoparticle Adlayer Properties by Photothermal Microscopy.

Many nanoparticle applications require molecular adlayers that impart desirable interfacial characteristics. Such characteristics are crucial in controlling the interaction of the nanoparticle with the environment or other nanoparticles; however, departures from bulk values are expected for adlayer properties and in situ methods to evaluate the magnitude of these departures, preferably on the scale of a single nanoparticle, are needed. Here we investigate the potential of single-particle photothermal microscopy for measuring the thermal properties of nanoparticle-supported, layer-by-layer grown polyelectrolytes.

Segmented GFP-like aptamer probes for functional imaging of viral genome trafficking.

Recently developed GFP-like RNA aptamers harbor a few unique potential benefits for in vivo RNA imaging applications, including co-packaging of viral genomes. Here we examine them in the context of co-packaging of RNA strands during virion assembly and trafficking. The approach is applicable both in vitro and in vivo, thus bridging an existing methodological gap.

Encapsulation of nanoparticles in virus protein shells.

The self-assembly of virus-like particles may lead to materials which combine the unique characteristics of viruses, such as precise size control and responsivity to environmental cues, with the properties of abiotic cargo. For a few different viruses, shell proteins are amenable to the in vitro encapsulation of non-genomic cargo in a regular protein cage. In this chapter we describe protocols of high-efficiency in vitro self-assembly around functionalized gold nanoparticles for three examples of icosahedral and non-icosahedral viral protein cages derived from a plant virus, an animal virus, and a human retrovirus.

Fusion of mApple and Venus fluorescent proteins to the Sindbis virus E2 protein leads to different cell-binding properties.

Fluorescent proteins (FPs) are widely used in real-time single virus particle studies to visualize, track and quantify the spatial and temporal parameters of viral pathways. However, potential functional differences between the wild type and the FP-tagged virus may specifically affect particular stages in the virus life-cycle. In this work, we genetically modified the E2 spike protein of Sindbis virus (SINV) with two FPs.

The packaging of different cargo into enveloped viral nanoparticles.

Viral nanoparticles used for biomedical applications must be able to discriminate between tumor or virus-infected host cells and healthy host cells. In addition, viral nanoparticles must have the flexibility to incorporate a wide range of cargo, from inorganic metals to mRNAs to small molecules. Alphaviruses are a family of enveloped viruses for which some species are intrinsically capable of systemic tumor targeting.

Magnetic virus-like nanoparticles in N. benthamiana plants: a new paradigm for environmental and agronomic biotechnological research.

This article demonstrates the encapsulation of cubic iron oxide nanoparticles (NPs) by Brome mosaic virus capsid shells and the formation, for the first time, of virus-based nanoparticles (VNPs) with cubic cores. Cubic iron oxide NPs functionalized with phospholipids containing poly(ethylene glycol) tails and terminal carboxyl groups exhibited exceptional relaxivity in magnetic resonance imaging experiments, which opens the way for in vivo MRI studies of systemic virus movement in plants. Preliminary data on cell-to-cell and long-distance transit behavior of cubic iron oxide NPs and VNPs in Nicotiana benthamiana leaves indicate that VNPs have specific transit properties, i.

Role of surface charge density in nanoparticle-templated assembly of bromovirus protein cages.

Self-assembling icosahedral protein cages have potentially useful physical and chemical characteristics for a variety of nanotechnology applications, ranging from therapeutic or diagnostic vectors to building blocks for hierarchical materials. For application-specific functional control of protein cage assemblies, a deeper understanding of the interaction between the protein cage and its payload is necessary. Protein-cage encapsulated nanoparticles, with their well-defined surface chemistry, allow for systematic control over key parameters of encapsulation such as the surface charge, hydrophobicity, and size.