Efficient encapsulation of functionalized spherical nanoparticles by viral protein cages was found to occur even if the nanoparticle is larger than the inner cavity of the native capsid. This result raises the intriguing possibility of reprogramming the self-assembly of viral structural proteins. The iron oxide nanotemplates used in this work are superparamagnetic, with a blocking temperature of about 250 K, making these virus-like particles interesting for applications such as magnetic resonance imaging and biomagnetic materials. Another novel feature of the virus-like particle assembly described in this work is the use of an anionic lipid micelle coat instead of a molecular layer covalently bound to the inorganic nanotemplate. Differences between the two functionalization strategies are discussed.

Self-assembling icosahedral protein cages have potencially 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. Independent control over these variables allows experimental testing of different assembly mechanism models. Previous studies done with Brome mosaic virus capsids and negatively charged gold nanoparticles indicated that the result of the selfassembly process depends on the diameter of the particle. However, in these experiments, the surface-ligand density was maintained at saturation levels, while the total charge and the radius of curvature remained coupled variables, making the interpretation of the observed dependence on the core size difficult. The current work furnishes evidence of a critical surface charge density for assembly through an analysis aimed at decoupling the surface charge and the core size.

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.

This article demonstrates the encapsulation of cubic iron oxide 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 nanoparticles (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 N. benthamiana leaves indicate that VNPs have specific transit properties, i.e., penetration into tissue and long-distance transfer through the vasculature in N. Benthamiana plants, even at low temperature (6° C), while NPs devoid of virus protein coats exhibit limited transport by comparison. These particles potentially open new opportunities for the high contrast functional imaging in plants and for the delivery of therapeutic anti-microbial cores into plants.

viruses, the CPs can self-assemble in vitro around a variety of non-genomic cargo, [6] forming shells that can be structurally identical to those of the wild type (wt) virus. However, wt virus assembly inside the heterogeneous, crowded environment of a host cell cytoplasm surprisingly leads to the large majority of virions being laden with genomic RNA. [7] The reason for non-specific encapsulation in vitro is relatively well understood. [5,8] The main driving force behind assembly at physiological ionic strengths are electrostatic interactions. [9] By comparison, the rate of empty capsids assembly, which can occur when electrostatic interactions are screened, is several orders of magnitude slower than co-assembly of coat proteins in presence of polyanionic species. [10] The question is then, how does a virus avoid production of virus-like particles that encapsulate many of the smaller, non-viral, transient RNAs and other polyions occurring in the cytoplasm? In attempting to answer this long-standing question we have studied the nature of chimeras which assemble out of small ssDNA oligonucleotides and viral coat protein. Cryo-electron microscopy (cryo-EM) and charge detection mass spectrometry (CDMS) analysis of in vitro assembly products suggest that CP shells do readily form around multiple oligonucleotides. However, these shells have specific, strained structures which easily split into large fragments. These may provide intermediates for correct, fast virion growth when cognate RNA, containing appropriate packaging signals becomes available. [11-13] 2. Results and Discussion In this work, virus-like particles (VLPs) were formed by mixing purified coat proteins of the brome mosaic virus (BMV) with two types of ssDNA oligonucleotides, both 52 nucleotides long. The two oligonucleotide fragments had different tertiary structures: the first one, hereafter called oligoB, originated from the BMV RNA genome sequence that interacts with the BMV CP N-terminal arm. [14,15] The second oligomer was a linear polyA polymer with no tertiary structure (see Figures S1 and S2, Supporting Information for assembly conditions, biochemical characterization, and transmission electron microscopy (TEM) characterization). To determine the masses of the VLPs formed, we employed charge detection mass spectrometry (CDMS). CDMS measures Non-enveloped RNA viruses pervade all domains of life. In a cell, they coassemble from viral RNA and capsid proteins. Virus-like particles can form in vitro where virtually any non-cognate polyanionic cargo can be packaged. How only viral RNA gets selected for packaging in vivo, in presence of myriad other polyanionic species, has been a puzzle. Through a combination of charge detection mass spectrometry and cryo-electron microscopy, it is determined that co-assembling brome mosaic virus (BMV) coat proteins and nucleic acid oligomers results in capsid structures and stoichiometries that differ from the icosahedral virion. These previously unknown shell structures are strained and less stable than the native ...

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 virus particle 28 nm diameter 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. This radiation brightening occurs only under short pulse excitation; steady-state excitation is characterized by conventional concentration quenching for any number of chromophores per particle. The observed suppression of fluorescence quenching is consistent with efficient, collective radiation at room temperature. Interestingly, radiation brightening disappears when the emitters spatial and/or dynamic heterogeneity is increased, suggesting that the template structural properties may play a role and opening a way towards novel, virus-enabled imaging vectors that have qualitatively different optical properties than state-of-the-art biophotonic agents. 1 arXiv:1907.00065v2 [physics.app-ph] 2 Jul 2019 Abbreviations VLP Keywords virus nanotechnology, directed assembly, biophotonics, nanolaser, sub-wavelength, superradiance, quantum coherence, fluorescence quenching Photoluminescence, particularly fluorescence, is used in a myriad of applications in which low-background, high spatial resolution, and rapid response are required for non-intrusive imaging, remote sensing, and control of transient chemical states. Thus, synthetic fluorophores are incorporated in sensors and detectors, e.g. for early warning of bio-aerosolthreats, used in operation rooms for intraoperative guidance in brain and prostate cancer surgery, 1 and in anti-counterfeiting materials. 2 Over the years spectacular improvements in chromophore photostability, wavelength range, biological integration, detectors and detection techniques, have pushed the limits of fluorescence imaging to realms never thought possible before. 3,4 Despite these improvements, and somewhat surprisingly in the context of ever more demanding cutting edge applications, fluorescence emission is still overwhelmingly by way of uncorrelated, random emission from multiple chromophores. Associated with this regime are undesirable characteristics such as self-quenching, when emitters are too close, and exponential decay that is relatively slow at molecular scale (typically, 1-5 ns). Extended excited state lifetimes limit emission brightness, increasing the likelihood of photobleaching, and making the quantum yield more prone to change in response to environmental fluctuations. 5 At the same time, a system's collective behavior can be much more than the sum of its

Abstract. 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). Both CCMV and BMV are approximately 4.6 megadalton (MDa) in mass, of which approximately 1 MDA originates from the genomic content of the virion. Both viruses are produced as mixtures of three particles carrying different segments of the genome, varying by approximately 0.1 MDA in mass (~2%). This mixture of particles poses a challenging analytical problem for high-resolution native MS analysis, given the large mass scales involved. We attempt to unravel the particle heterogeneity using both Q-TOF and Orbitrap mass spectrometers extensively modified for analysis of very large assemblies. We show that manipulation of the charging behavior can provide assistance in assigning the correct charge states. Despite their challenging size and heterogeneity, we obtained native mass spectra with resolved series of charge states for both BMV and CCMV, demonstrating that native MS of endogenous multipartite virions is feasible.

Viral nanoparticles (VNPs) are becoming versatile tools in platform technology development. Their well-defined structures as well as their programmability through chemical and genetic modification allow VNPs to be engineered for potential imaging and therapeutic applications. In this article, we report the application of a variety of bioconjugation chemistries to the plant VNP Brome mosaic virus (BMV). Functional BMV nanoparticles displaying multiple copies of fluorescent dyes, PEG molecules, chemotherapeutic drug moieties, targeting proteins and cell penetrating peptides were formulated. This opens the door for the application of BMV in nanomedicine.