The spectrum of derived Mackey functors

Peer reviewedPostprin

Genome organization and interaction with capsid protein in a multipartite RNA virus.

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content-specifically, RNAs 3 and 4-assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific "packaging signals" throughout the viral RNA to package their monopartite genomes

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.Engineering and Applied SciencesPhysic

Effect of secondary structure on the size, configurational statistics, and packaging of long-RNA by viral capsid protein

Many viruses use long (thousands of nucleotides) single-stranded (ss)RNAs as their genomic material. Such viruses can be as simple as a single RNA molecule encapsidated inside a shell (capsid) composed of many copies of a single capsid protein (CP). Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV), the two sibling viruses studied throughout this work, are model ssRNA viruses that are capable of spontaneous self-assembly \textit{in vitro}. This work aims to elucidate how RNA content affects both the structure of the particles formed and their physical properties. In particular, particles containing one or another of a variety of RNA molecules are compared using physical and biological tools. The results of this work further our understanding of the self-assembly of ssRNA viruses, and the physical forces that result in their structure and dynamical properties.Chapter one is an introductory chapter, introducing the reader to viruses, ssRNAs and their secondary/tertiary structures, CCMV and BMV, and the \textit{in vitro} self-assembly of these viruses. Chapters two and three discuss the production of viral-length polyU, an RNA molecule that lacks secondary structure due to its inability to base-pair or base-stack, and the packaging of this unique, structureless RNA with CCMV CP. Chapter four discusses the cryo-electron microscopy (cryoEM) asymmetric reconstruction of BMV virions, which allows for unprecedented visualization of the RNA genome inside a multipartite virus--a virus with a genome comprised of more than one RNA packaged into several indistinguishable particles. Chapter five summarizes the results of this work, and provides some future experiments related to the work discussed herein

Effect of secondary structure on the size, configurational statistics, and packaging of long-RNA by viral capsid protein

Many viruses use long (thousands of nucleotides) single-stranded (ss)RNAs as their genomic material. Such viruses can be as simple as a single RNA molecule encapsidated inside a shell (capsid) composed of many copies of a single capsid protein (CP). Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV), the two sibling viruses studied throughout this work, are model ssRNA viruses that are capable of spontaneous self-assembly \textit{in vitro}. This work aims to elucidate how RNA content affects both the structure of the particles formed and their physical properties. In particular, particles containing one or another of a variety of RNA molecules are compared using physical and biological tools. The results of this work further our understanding of the self-assembly of ssRNA viruses, and the physical forces that result in their structure and dynamical properties.Chapter one is an introductory chapter, introducing the reader to viruses, ssRNAs and their secondary/tertiary structures, CCMV and BMV, and the \textit{in vitro} self-assembly of these viruses. Chapters two and three discuss the production of viral-length polyU, an RNA molecule that lacks secondary structure due to its inability to base-pair or base-stack, and the packaging of this unique, structureless RNA with CCMV CP. Chapter four discusses the cryo-electron microscopy (cryoEM) asymmetric reconstruction of BMV virions, which allows for unprecedented visualization of the RNA genome inside a multipartite virus--a virus with a genome comprised of more than one RNA packaged into several indistinguishable particles. Chapter five summarizes the results of this work, and provides some future experiments related to the work discussed herein

The spectrum of derived Mackey functors

Peer reviewedPostprin

The Effect of RNA Secondary Structure on the Self-Assembly of Viral Capsids

Previous work has shown that purified capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) is capable of packaging both purified single-stranded RNA molecules of normal composition (comparable numbers of A, U, G, and C nucleobases) and of varying length and sequence, and anionic synthetic polymers such as polystyrene sulfonate. We find that CCMV CP is also capable of packaging polyU RNAs, which-unlike normal-composition RNAs-do not form secondary structures and which act as essentially structureless linear polymers. Following our canonical two-step assembly protocol, polyU RNAs ranging in length from 1000 to 9000 nucleotides (nt) are completely packaged. Surprisingly, negative-stain electron microscopy shows that all lengths of polyU are packaged into 22-nm-diameter particles despite the fact that CCMV CP prefers to form 28-nm-diameter (T = 3) particles when packaging normal-composition RNAs. PolyU RNAs >5000 nt in length are packaged into multiplet capsids, in which a single RNA molecule is shared between two or more 22-nm-diameter capsids, in analogy with the multiplets of 28-nm-diameter particles formed with normal-composition RNAs >5000 nt long. Experiments in which viral RNA competes for viral CP with polyUs of equal length show that polyU, despite its lack of secondary structure, is packaged more efficiently than viral RNA. These findings illustrate that the secondary structure of the RNA molecule-and its absence-plays an essential role in determining capsid structure during the self-assembly of CCMV-like particles

Rna Compaction in the Presence of Polyvalent Cations

The effects of polyvalent cations on the effective size and charge of double-stranded DNA (dsDNA) have been well studied. In the presence of polyvalent cations, dsDNA in dilute solution undergoes a single-molecule, first-order phase transition, otherwise called condensation: more explicitly, upon onset of 90% neutralization of the phosphate backbone, the DNA undergoes discontinuous compaction into tightly wound toroids. However, the effects of these cations on long single-stranded RNAs (ssRNA) have not been well characterized. In this study we use centrifugation methods to examine the effective size of long ssRNAs in solutions of increasing concentration of the tetravalent cation spermine. In contrast to the case of dsDNA, we find only a continuous decrease in the size of ssRNA upon increase in spermine concentration. However, the decrease is significant enough to suggest that RNA molecules longer than viral genomes can be packaged in vitro into virus-like vectors for gene delivery

Genome organization and interaction with capsid protein in a multipartite RNA virus.

We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content-specifically, RNAs 3 and 4-assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific "packaging signals" throughout the viral RNA to package their monopartite genomes