<i>In Singulo</i> Probing of Viral RNA Dynamics by Multichromophore Fluorescence Dequenching

Current understanding of virus life-cycle states and transitions between them is mainly built on knowledge of the protein shell structure encapsulating the genome. Little is known about the genome fate during viral transitions. Here, changes in the fluorescence rate from multilabeled transcript viral RNAs encapsulated in Brome mosaic virus capsids were examined as a function of the RNA state. A simple kinetic model relating chain compactness to single-molecule fluorescence emission suggests that in a dense multichromophore system the rate of energy transfer should scale with distance more gradually than the rate of the Förster energy transfer between two chromophores, which varies sharply as the reciprocal of distance to the sixth power. As a proof-of-principle experiment, we have compared predictions from a numerical model for confined diffusive motion with the fluorescence emission from virus-encapsulated and free single RNA molecules decorated with multiple cyanine dyes and encapsulated inside microscopic emulsion droplets. We found that the effective quantum yield per labeled particle depends on the expansion state, in agreement with theoretical predictions. Since fluorescence single particle tracking is now a well-established methodology for the study of virus life cycle, the findings reported here may pave the way toward reducing the existing gap between <i>in vitro</i> and cellular <i>in singulo</i> studies of the fates of viral RNA

Budding Pathway in the Templated Assembly of Viruslike Particles

A new pathway for the assembly of viral capsid protein around inorganic nanoparticle cores was observed by time-course light scattering and cryo-electron tomography. Gold nanoparticles with an average diameter of 11.3 nm have been used as a template for the assembly of Brome mosaic virus (BMV) capsid protein at different concentrations. At least at low protein concentrations the kinetic features of the scattering and extinction measurements are consistent with the initial rapid formation of large nanoparticle–protein clusters, which subsequently separate into individual viruslike particles (VLPs). The occurrence of multiparticle clusters at short times after mixing nanoparticles and proteins was confirmed by cryo-EM. Cryo-electron tomography of the multiparticle clusters yielded an average surface-to-surface interparticle distance of ∼7.5 nm, equivalent to ∼1.5 times the thickness of a protein shell. We propose a scenario in which VLP generation may take place through monomer exchange between aggregated particles with defect-ridden or incomplete shells, leading to the formation of stable icosahedral shells, which eventually bud off the aggregate. Together with results from previous works, the findings highlight the astonishing versatility of plant virus capsid protein assembly. This previously unknown mechanism for VLP formation has features that may have relevance for the crowded environment characterizing virus factories in the cell

Probing the Link among Genomic Cargo, Contact Mechanics, and Nanoindentation in Recombinant Adeno-Associated Virus 2

Recombinant adeno-associated virus (AAV) is a promising gene therapy vector. To make progress in this direction, the relationship between the characteristics of the genomic cargo and the capsid stability must be understood in detail. The goal of this study is to determine the role of the packaged vector genome in the response of AAV particles to mechanical compression and adhesion to a substrate. Specifically, we used atomic force microscopy to compare the mechanical properties of empty AAV serotype 2 (AAV2) capsids and AAV2 vectors packaging single-stranded DNA or self-complementary DNA. We found that all species underwent partial deformation upon adsorption from buffer on an atomically flat graphite surface. Upon adsorption, a preferred orientation toward the twofold symmetry axis on the capsid, relative to the substrate, was observed. The magnitude of the bias depended on the cargo type, indicating that the interfacial properties may be influenced by cargo. All particles showed a significant relative strain before rupture. Different from interfacial interactions, which were clearly cargo-dependent, the elastic response to directional stress was largely dominated by the capsid properties. Nevertheless, small differences between particles laden with different cargo were measurable; scAAV vectors were the most resilient to external compression. We also show how elastic constant and rupture force data sets can be analyzed according a multivariate conditional probability approach to determine the genome content on the basis of a database of mechanical properties acquired from nanoindentation assays. Implications for understanding how recombinant AAV capsid–genome interactions can affect vector stability and effectiveness of gene therapy applications are discussed

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. We show that nanometer changes in adlayer thickness can be detected this way, and the water content of the nanoparticle-supported adlayers can be estimated

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. Comparison of chiral angles and packing defects observed by <i>in situ</i> atomic force microscopy with those obtained from molecular dynamics models offers insight into the mechanism of growth, and the influence of stresses associated with intrinsic curvature and assembly pathways

Structure versus Composition: A Single-Particle Investigation of Plasmonic Bimetallic Nanocrystals

Stellated bimetallic nanostructures are a new class of plasmonic colloids in which the interplay between composition and overall architecture can provide tunable optical properties and new functionality. However, decoupling the complex compositional and structural contributions to the localized surface plasmon resonance (LSPR) remains a challenge, especially when the monometallic counterparts are not synthetically accessible for comparison. Here, stellated Au–Pd nanocrystals (NCs) with <i>O</i>_<i>h</i> symmetry are used as a model system to decouple structural and complex compositional effects on LSPR. Single-particle correlation of the LSPR with the structure of octopodal Au–Pd NCs was achieved using optical dark-field spectroscopy with scanning electron microscopy. These measurements were compared to calculations of the optical properties of structurally similar Au-only octopods by the finite difference time domain method. This comparison enabled the role of complex composition, which was determined by scanning transmission electron microscopy–energy-dispersive spectrometry measurements, on the LSPR to be elucidated from the structural contributions. This methodology provides a powerful framework to guide the design of new plasmonic colloids through both structure and composition

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. By numerical simulations, we examine how these changes depend on the symmetry and extent of the deformation and on both the propagation direction and polarization of the incident radiation. Such a sensor could prove useful in studies of the mechanisms of nanoparticle or virus translocation in the confines of a host cell

Physiochemical Properties of <i>Caulobacter crescentus</i> Holdfast: A Localized Bacterial Adhesive

To colonize surfaces, the bacterium <i>Caulobacter crescentus</i> employs a polar polysaccharide, the holdfast, located at the end of a thin, long stalk protruding from the cell body. Unlike many other bacteria which adhere through an extended extracellular polymeric network, the holdfast footprint area is tens of thousands times smaller than that of the total bacterium cross-sectional surface, making for some very demanding adhesion requirements. At present, the mechanism of holdfast adhesion remains poorly understood. We explore it here along three lines of investigation: (a) the impact of environmental conditions on holdfast binding affinity, (b) adhesion kinetics by dynamic force spectroscopy, and (c) kinetic modeling of the attachment process to interpret the observed time-dependence of the adhesion force at short and long time scales. A picture emerged in which discrete molecular units called adhesins are responsible for initial holdfast adhesion, by acting in a cooperative manner

Physiochemical Properties of <i>Caulobacter crescentus</i> Holdfast: A Localized Bacterial Adhesive

To colonize surfaces, the bacterium <i>Caulobacter crescentus</i> employs a polar polysaccharide, the holdfast, located at the end of a thin, long stalk protruding from the cell body. Unlike many other bacteria which adhere through an extended extracellular polymeric network, the holdfast footprint area is tens of thousands times smaller than that of the total bacterium cross-sectional surface, making for some very demanding adhesion requirements. At present, the mechanism of holdfast adhesion remains poorly understood. We explore it here along three lines of investigation: (a) the impact of environmental conditions on holdfast binding affinity, (b) adhesion kinetics by dynamic force spectroscopy, and (c) kinetic modeling of the attachment process to interpret the observed time-dependence of the adhesion force at short and long time scales. A picture emerged in which discrete molecular units called adhesins are responsible for initial holdfast adhesion, by acting in a cooperative manner

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. Alphavirus virus-like particles, or viral nanoparticles, can be generated from <i>in vitro</i> self-assembled core-like particles using nonviral nucleic acid. In this work, we expand on the types of cargo that can be incorporated into alphavirus core-like particles and the molecular requirements for packaging this cargo. We demonstrate that different core-like particle templates can be further enveloped to form viral nanoparticles that are capable of cell entry. We propose that alphaviruses can be selectively modified to create viral nanoparticles for biomedical applications and basic research