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. We mix coat protein and RNA from bacteriophage MS2, and we use a combination of gel electrophoresis, dynamic light scattering, and transmission electron microscopy to investigate the assembly products. We show that with increasing coat-protein concentration, the products transition from well-formed MS2 VLPs to ``monster'' particles consisting of multiple partial capsids to RNA-protein condensates consisting of large networks of RNA and partially assembled capsids. We argue that the transition from well-formed to monster particles arises because the assembly follows a nucleation-and-growth pathway in which the nucleation rate depends sensitively on the coat-protein concentration, such that at high protein concentrations, multiple nuclei can form on each RNA strand. To understand the formation of the condensates, which occurs at even higher coat-protein concentrations, we use Monte Carlo simulations with coarse-grained models of capsomers and RNA. These simulations suggest that the the formation of condensates occurs by the adsorption of protein to the RNA followed by the assembly of capsids. Multiple RNA molecules can become trapped when a capsid grows from capsomers attached to two different RNA molecules or when excess protein bridges together growing capsids on different RNA molecules. Our results provide insight into an important biophysical process and could inform design rules for making VLPs for various applications.

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. We mix coat protein and RNA from bacteriophage MS2, and we use a combination of gel electrophoresis, dynamic light scattering, and transmission electron microscopy to investigate the assembly products. We show that with increasing coat-protein concentration, the products transition from well-formed MS2 VLPs to "monster" particles consisting of multiple partial capsids to RNA-protein condensates consisting of large networks of RNA and partially assembled capsids. We argue that the transition from well-formed to monster particles arises because the assembly follows a nucleation-and-growth pathway in which the nucleation rate depends sensitively on the coat-protein concentration, such that at high protein concentrations, multiple nuclei can form on each RNA strand. To understand the formation of the condensates, which occurs at even higher coat-protein concentrations, we use Monte Carlo simulations with coarse-grained models of capsomers and RNA. These simulations suggest that the formation of condensates occurs by the adsorption of protein to the RNA followed by the assembly of capsids. Multiple RNA molecules can become trapped when a capsid grows from capsomers attached to two different RNA molecules or when excess protein bridges together growing capsids on different RNA molecules. Our results provide insight into an important biophysical process and could inform design rules for making VLPs for various applications.

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. This approach yields best-fit parameters, including the particle's three-dimensional position and polarizability, as well as uncertainties and correlations between these parameters. Building on recent work, we develop a model that is parameterized for rapid fitting. The model is designed to work with Hamiltonian Monte Carlo techniques that leverage automatic differentiation. We validate this approach by fitting the model to interferometric images of colloidal nanoparticles. We apply the method to track a diffusing particle in three dimensions, to directly infer the diffusion coefficient of a nanoparticle without calculating a mean-square displacement, and to quantify the ejection of DNA from an individual lambda phage virus, demonstrating that the approach can be used to infer both static and dynamic properties of nanoscale systems.

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. Here, we used transmission electron microscopy (TEM) and cryogenic electron microscopy (Cryo-EM) to address some knowledge gaps. We concluded that mature WSSV virions with a stout oval-like shape do not have tail-like extensions. Furthermore, there were two distinct ends in WSSV nucleocapsids: a portal cap and a closed base. A C14 symmetric structure of the WSSV nucleocapsid was also proposed, according to our Cryo-EM map. Immunoelectron microscopy (IEM) revealed that VP664 proteins, the main components of the 14 assembly units, form a ring-like architecture. Moreover, WSSV nucleocapsids were also observed to undergo unique helical dissociation. Based on these new results, we propose a novel morphogenetic pathway of WSSV.

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. We validate this patch-probe method using the 1,058-nucleotide RNA genome of satellite tobacco mosaic virus (STMV), which has previously been shown to have multiple long-range connections. Our results not only indicate long duplexes that agree with previous structures but also reveal the prevalence of competing connections. Together, these results suggest that globally-folded and locally-folded structures coexist in solution. We show that the prevalence of connections changes when pseudouridine, an important component of natural and synthetic RNA molecules, is substituted for uridine in STMV RNA.

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. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed, but the time to grow a capsid after nucleation is largely unaffected. These results suggest that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intraprotein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses.

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. Specifically, we successfully measure the diameter of every gold nanosphere in an ensemble that consists of several hundreds of 40 nm particles, with an individual precision below 17% (±8 nm). In addition, we confirm the relevance of our approach with respect to bioanalytics by analyzing 70 nm λ-phages. Overall these features, together with the strongly reduced demand for memory space, principally allows us to record thousands of frames and to achieve high frame rates for high precision tracking of nanoscale objects.

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. By recording how many coat proteins bind to each of many individual RNA strands, we find that assembly proceeds by nucleation followed by monotonic growth. Our measurements reveal the assembly pathways in quantitative detail and also show their failure modes. We use these results to critically examine models of the assembly process.

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. Additionally, we point out that separating the assembly reaction into multiple steps-by successively lowering the ionic strength and then the pH of the assembly buffers-results in the highest yields of well-formed, nuclease-resistant, CCMV-like particles. Finally, we describe methods for characterizing the assembly products using native agarose gel electrophoresis and negative-stain transmission electron microscopy.

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. coli holograms can yield quantitative information about the bacterium's body rotation and tumbles, offering a precise way to track fine details of bacterial motility. We are able to extract the cell's three-dimensional (3D) position and orientation and recover behavior such as body angle rotation during runs, tumbles, and pole reversal. Our technique is label-free and capable of frame rates limited only by the camera.

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. The technique determines the in-plane motion of the phage to nanometer precision, and the height of the phage above the surface to 100 nm precision. Additionally, we track the DNA content of individual phages as they eject their genome following the addition of detergent-solubilized LamB receptor. The technique determines the fraction of DNA remaining in the phage to within 10% of the total 48.5 kilobase pairs. Analysis of the data reveals that under certain conditions, λ phages move along the surface with their heads down and intermittently stick to the surface by their tails, causing them to stand up. Furthermore, we find that in buffer containing high concentrations of both monovalent and divalent salts, λ phages eject their entire DNA in about 7 s. Taken together, these measurements highlight the potential of holographic microscopy to resolve the fast kinetics of the early stages of phage infection.

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., multiples of 60) copies of a single protein. Indeed, the smallest viral genomes include essentially only two genes, on the order of a thousand times fewer than the next-simplest organisms like bacteria and yeast. For these reasons, it is possible and even fruitful to take a reductionist approach to viruses and to understand how they work in terms of fundamental physical principles. In this Account, we discuss our recent physical chemistry approach to studying the self-assembly of a particular spherical virus (cowpea chlorotic mottle virus) whose reconstitution from RNA and capsid protein has long served as a model for virus assembly. While previous studies have clarified the roles of certain physical (electrostatic, hydrophobic, steric) interactions in the stability and structure of the final virus, it has been difficult to probe these interactions during assembly because of the inherently short lifetimes of the intermediate states. We feature the role of pH in tuning the magnitude of the interactions among capsid proteins during assembly: in particular, by making the interactions between proteins sufficiently weak, we are able to stall the assembly process and interrogate the structure and composition of particular on-pathway intermediates. Further, we find that the strength of the lateral attractions between RNA-bound proteins plays a key role in addressing several outstanding questions about assembly: What determines the pathway or pathways of assembly? What is the importance of kinetic traps and hysteresis? How do viruses copackage multiple short (compared with wild-type) RNAs or single long RNAs? What determines the relative packaging efficiencies of different RNAs when they are forced to compete for an insufficient supply of protein? And what is the limit on the length of RNA that can be packaged by CCMV capsid protein?

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. Here we present a tracking method based on elastic light scattering that enables long-duration measurements of nanoparticle dynamics at rates of thousands of frames per second. We contain the particles within a single-mode silica fiber having a subwavelength, nanofluidic channel and illuminate them using the fiber's strongly confined optical mode. The diffusing particles in this cylindrical geometry are continuously illuminated inside the collection focal plane. We show that the method can track unlabeled dielectric particles as small as 20 nm as well as individual cowpea chlorotic mottle virus (CCMV) virions-26 nm in size and 4.6 megadaltons in mass-at rates of over 3 kHz for durations of tens of seconds. Our setup is easily incorporated into common optical microscopes and extends their detection range to nanometer-scale particles and macromolecules. The ease-of-use and performance of this technique support its potential for widespread applications in medical diagnostics and micro total analysis systems.

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. But, before capsids are formed, the binding of protein remains reversible and introduction of another RNA species-with a different length and/or sequence-is found experimentally to result in significant redistribution of protein. Here we argue that, for a given RNA mass, the sequence with the highest affinity for protein is the one with the most compact secondary structure arising from self-complementarity; similarly, a long RNA steals protein from an equal mass of shorter ones. In both cases, it is the lateral attractions between bound proteins that determines the relative CP affinities of the RNA templates, even though the individual binding sites are identical. We demonstrate this with Monte Carlo simulations, generalizing the Rosenbluth method for excluded-volume polymers to include branching of the polymers and their reversible binding by protein.

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. Our synthesis does not involve covalent chemistry of any kind; rather, we trigger capsid assembly on a scaffold of viral RNA that is hybridized at one end to a complementary DNA strand. Interaction of CCMV capsid protein with this RNA-DNA template leads to selective packaging of the RNA portion into a well-formed capsid but leaves the hybridized portion poking out of the capsid through a small hole. We show that the nucleic acid protruding from the capsid is capable of binding free DNA strands and DNA-functionalized colloidal particles. Separately, we show that the RNA-DNA scaffold can be used to nucleate virus formation on a DNA-functionalized surface. We believe this self-assembly strategy can be adapted to viruses other than CCMV.

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. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures.

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. The main focus of this review is to (i) apprise readers of the technical breakthroughs that have facilitated the dissection of replication from virion assembly and genome packaging in vivo and (ii) describe the critical factors that have been shown to be involved in the regulation and integration of these processes.

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

UNLABELLED: We have recently discovered (R. D. Cadena-Nava et al., J. Virol. 86:3318-3326, 2012, doi:10.1128/JVI.06566-11) that the in vitro packaging of RNA by the capsid protein (CP) of cowpea chlorotic mottle virus is optimal when there is a significant excess of CP, specifically that complete packaging of all of the RNA in solution requires sufficient CP to provide charge matching of the N-terminal positively charged arginine-rich motifs (ARMS) of the CPs with the negatively charged phosphate backbone of the RNA. We show here that packaging results from the initial formation of a charge-matched protocapsid consisting of RNA decorated by a disordered arrangement of CPs. This protocapsid reorganizes into the final, icosahedrally symmetric nucleocapsid by displacing the excess CPs from the RNA to the exterior surface of the emerging capsid through electrostatic attraction between the ARMs of the excess CP and the negative charge density of the capsid exterior. As a test of this scenario, we prepare CP mutants with extra and missing (relative to the wild type) cationic residues and show that a correspondingly smaller and larger excess, respectively, of CP is needed for complete packaging of RNA. IMPORTANCE: Cowpea chlorotic mottle virus (CCMV) has long been studied as a model system for the assembly of single-stranded RNA viruses. While much is known about the electrostatic interactions within the CCMV virion, relatively little is known about these interactions during assembly, i.e., within intermediate states preceding the final nucleocapsid structure. Theoretical models and coarse-grained molecular dynamics simulations suggest that viruses like CCMV assemble by the bulk adsorption of CPs onto the RNA driven by electrostatic attraction, followed by structural reorganization into the final capsid. Such a mechanism facilitates assembly by condensing the RNA for packaging while simultaneously concentrating the local density of CP for capsid nucleation. We provide experimental evidence of such a mechanism by demonstrating that efficient assembly is initiated by the formation of a disordered protocapsid complex whose stoichiometry is governed by electrostatics (charge matching of the anionic RNA and the cationic N termini of the CP).

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. We show that the degree of cooperativity of virus assembly depends not only on the relative strength of the CP-CP and CP-RNA interactions but also on the RNA being short: a 500-nt RNA molecule cannot form a capsid by itself, so its packaging requires the aggregation of multiple CP-RNA complexes. By using fluorescence correlation spectroscopy (FCS), we show that at neutral pH and sufficiently low concentrations RNA and CP form complexes that are smaller than the wild-type capsid and that four 500-nt RNAs are packaged into virus-like particles (VLPs) only upon lowering the pH. Further, a variety of bulk-solution techniques confirm that fully ordered VLPs are formed only upon acidification. On the basis of these results, we argue that the observed high degree of cooperativity involves equilibrium between multiple CP/RNA complexes.