Effect of coat-protein concentration on the self-assembly of bacteriophage MS2 capsids around RNA.

Self-assembly is a vital part of the life cycle of certain icosahedral RNA viruses. Furthermore, the assembly process can be harnessed to make icosahedral virus-like particles (VLPs) from coat protein and RNA in vitro. Although much previous work has explored the effects of RNA-protein interactions on the assembly products, relatively little research has explored the effects of coat-protein concentration.

Effect of coat-protein concentration on the self-assembly of bacteriophage MS2 capsids around RNA.

Self-assembly is a vital part of the life cycle of certain icosahedral RNA viruses. Furthermore, the assembly process can be harnessed to make icosahedral virus-like particles (VLPs) from coat protein and RNA . Although much previous work has explored the effects of RNA-protein interactions on the assembly products, relatively little research has explored the effects of coat-protein concentration.

Precise characterization of nanometer-scale systems using interferometric scattering microscopy and Bayesian analysis.

Interferometric scattering microscopy can image the dynamics of nanometer-scale systems. The typical approach to analyzing interferometric images involves intensive processing, which discards data and limits the precision of measurements. We demonstrate an alternative approach: modeling the interferometric point spread function and fitting this model to data within a Bayesian framework.

Multiple Nucleocapsid Structural Forms of Shrimp White Spot Syndrome Virus Suggests a Novel Viral Morphogenetic Pathway.

White spot syndrome virus (WSSV) is a very large dsDNA virus. The accepted shape of the WSSV virion has been as ellipsoidal, with a tail-like extension. However, due to the scarcity of reliable references, the pathogenesis and morphogenesis of WSSV are not well understood.

Measuring intramolecular connectivity in long RNA molecules using two-dimensional DNA patch-probe arrays.

We describe a simple method to infer intramolecular connections in a population of long RNA molecules in vitro. First we add DNA oligonucleotide "patches" that perturb the RNA connections, then we use a microarray containing a complete set of DNA oligonucleotide "probes" to record where perturbations occur. The pattern of perturbations reveals couplings between different regions of the RNA sequence, from which we infer connections as well as their prevalences in the population.

Single-particle studies of the effects of RNA-protein interactions on the self-assembly of RNA virus particles.

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of more than 500 individual particles of brome mosaic virus (BMV)-for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer.

Tracking and Analyzing the Brownian Motion of Nano-objects Inside Hollow Core Fibers.

Tracking and analyzing the individual diffusion of nanoscale objects such as proteins and viruses is an important methodology in life science. Here, we show a sensor that combines the efficiency of light line illumination with the advantages of fluidic confinement. Tracking of freely diffusing nano-objects inside water-filled hollow core fibers with core diameters of tens of micrometers using elastically scattered light from the core mode allows retrieving information about the Brownian motion and the size of each particle of the investigated ensemble individually using standard tracking algorithms and the mean squared displacement analysis.

Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome.

Self-assembly is widely used by biological systems to build functional nanostructures, such as the protein capsids of RNA viruses. But because assembly is a collective phenomenon involving many weakly interacting subunits and a broad range of timescales, measurements of the assembly pathways have been elusive. We use interferometric scattering microscopy to measure the assembly kinetics of individual MS2 bacteriophage capsids around MS2 RNA.

Protocol for Efficient Cell-Free Synthesis of Cowpea Chlorotic Mottle Virus-Like Particles Containing Heterologous RNAs.

We report a protocol for efficient cell-free synthesis of cowpea chlorotic mottle virus (CCMV)-like particles containing a broad range of lengths and sequences of RNA. Our protocol starts with a purified stock of wild-type CCMV (protocols for harvesting and purifying the virus are detailed elsewhere) and features three basic steps: disassembly of the CCMV and purification of the capsid protein (CP) from the viral RNA; coassembly of the purified CP and an RNA of choice; and characterization of the assembly products. We highlight several key factors that increase the yield of the assembly reaction: the CP should be uncleaved and sufficiently free of viral RNA; the length of the RNA should be between about 100 and 4000 nucleotides; and the stoichiometry of CP and RNA should be 6-1 by mass.

Tracking E. coli runs and tumbles with scattering solutions and digital holographic microscopy.

We use in-line digital holographic microscopy to image freely swimming E. coli. We show that fitting a light scattering model to E.

Dynamic Measurements of the Position, Orientation, and DNA Content of Individual Unlabeled Bacteriophages.

A complete understanding of the cellular pathways involved in viral infections will ultimately require a diverse arsenal of experimental techniques, including methods for tracking individual viruses and their interactions with the host. Here we demonstrate the use of holographic microscopy to track the position, orientation, and DNA content of unlabeled bacteriophages (phages) in solution near a planar, functionalized glass surface. We simultaneously track over 100 individual λ phages at a rate of 100 Hz across a 33 μm × 33 μm portion of the surface.

Physical Principles in the Self-Assembly of a Simple Spherical Virus.

Viruses are unique among living organisms insofar as they can be reconstituted "from scratch", that is, synthesized from purified components. In the simplest cases, their "parts list" numbers only two: a single molecule of nucleic acid and many (but a very special number, i.e.

Fast, Label-Free Tracking of Single Viruses and Weakly Scattering Nanoparticles in a Nanofluidic Optical Fiber.

High-speed tracking of single particles is a gateway to understanding physical, chemical, and biological processes at the nanoscale. It is also a major experimental challenge, particularly for small, nanometer-scale particles. Although methods such as confocal or fluorescence microscopy offer both high spatial resolution and high signal-to-background ratios, the fluorescence emission lifetime limits the measurement speed, while photobleaching and thermal diffusion limit the duration of measurements.

Role of RNA Branchedness in the Competition for Viral Capsid Proteins.

To optimize binding-and packaging-by their capsid proteins (CP), single-stranded (ss) RNA viral genomes often have local secondary/tertiary structures with high CP affinity, with these "packaging signals" serving as heterogeneous nucleation sites for the formation of capsids. Under typical in vitro self-assembly conditions, however, and in particular for the case of many ssRNA viruses whose CP have cationic N-termini, the adsorption of CP by RNA is nonspecific because the CP concentration exceeds the largest dissociation constant for CP-RNA binding. Consequently, the RNA is saturated by bound protein before lateral interactions between CP drive the homogeneous nucleation of capsids.

A Simple RNA-DNA Scaffold Templates the Assembly of Monofunctional Virus-Like Particles.

Using the components of a particularly well-studied plant virus, cowpea chlorotic mottle virus (CCMV), we demonstrate the synthesis of virus-like particles (VLPs) with one end of the packaged RNA extending out of the capsid and into the surrounding solution. This construct breaks the otherwise perfect symmetry of the capsid and provides a straightforward route for monofunctionalizing VLPs using the principles of DNA nanotechnology. It also allows physical manipulation of the packaged RNA, a previously inaccessible part of the viral architecture.

Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy.

The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms.

Integration of replication and assembly of infectious virions in plant RNA viruses.

For all plant pathogenic viruses with positive-strand RNA genomes, the assembly of infectious virions is a carefully orchestrated process. The mature virions of such viruses exhibit a remarkable degree of packaging specificity, despite the opportunity that exists to package cellular RNAs. Recent technical developments in the fields of molecular and cellular biology have revealed that the processes regulating genome replication and virion assembly are integrated.

Role of electrostatics in the assembly pathway of a single-stranded RNA virus.

Unlabelled: We have recently discovered (R. D. Cadena-Nava et al.

Characterization of Viral Capsid Protein Self-Assembly around Short Single-Stranded RNA.

For many viruses, the packaging of a single-stranded RNA (ss-RNA) genome is spontaneous, driven by capsid protein-capsid protein (CP) and CP-RNA interactions. Furthermore, for some multipartite ss-RNA viruses, copackaging of two or more RNA molecules is a common strategy. Here we focus on RNA copackaging in vitro by using cowpea chlorotic mottle virus (CCMV) CP and an RNA molecule that is short (500 nucleotides (nts)) compared to the lengths (≈3000 nts) packaged in wild-type virions.

The assembly pathway of an icosahedral single-stranded RNA virus depends on the strength of inter-subunit attractions.

The strength of attraction between capsid proteins (CPs) of cowpea chlorotic mottle virus (CCMV) is controlled by the solution pH. Additionally, the strength of attraction between CP and the single-stranded RNA viral genome is controlled by ionic strength. By exploiting these properties, we are able to control and monitor the in vitro co-assembly of CCMV CP and single-stranded RNA as a function of the strength of CP-CP and CP-RNA attractions.

Reconstituted plant viral capsids can release genes to mammalian cells.

The nucleocapsids of many plant viruses are significantly more robust and protective of their RNA contents than those of enveloped animal viruses. In particular, the capsid protein (CP) of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is of special interest because it has been shown to spontaneously package, with high efficiency, a large range of lengths and sequences of single-stranded RNA molecules. In this work we demonstrate that hybrid virus-like particles, assembled in vitro from CCMV CP and a heterologous RNA derived from a mammalian virus (Sindbis), are capable of releasing their RNA in the cytoplasm of mammalian cells.

Self-assembly of viral capsid protein and RNA molecules of different sizes: requirement for a specific high protein/RNA mass ratio.

Virus-like particles can be formed by self-assembly of capsid protein (CP) with RNA molecules of increasing length. If the protein "insisted" on a single radius of curvature, the capsids would be identical in size, independent of RNA length. However, there would be a limit to length of the RNA, and one would not expect RNA much shorter than native viral RNA to be packaged unless multiple copies were packaged.

Exploiting fluorescent polymers to probe the self-assembly of virus-like particles.

The inside surfaces of the protein shells of many viruses are positively charged, thereby enhancing the self-assembly of capsid proteins around their (oppositely charged) RNA genome. These proteins have been shown to organize similarly around a variety of nonbiological, negatively charged, polymers, for example, poly(styrene sulfonate) (PSS), forming virus-like particles (VLPs). We have demonstrated recently that the VLPs formed from cowpea chlorotic mottle virus (CCMV) capsid protein increase in size (from T=2 to T=3 structures) upon increase in PSS molecular weight (from 400 kDa to 3.