[We began a previous research summary with the phrase "Certainly one of the most intriguing facts about viruses is that", but we nevertheless begin this one that way as well, because we find that almost every aspect of virus formation is remarkable.]
Certainly one of the most intriguing facts about viruses is that they can be reconstituted in vitro from purified components, i.e., synthesized "from scratch" in the lab! More explicitly, as long ago as 1955, virions of Tobacco Mosaic Virus (TMV) were made by mixing its capsid protein and its RNA genome in aqueous solution; these particles were shown to be fully infectious, and indistinguishable from the virions purified from infected host cells. Similarly, in 1967, infectious virions of Cowpea Chlorotic Mottle Virus (CCMV) were synthesized from its capsid protein and RNA. Unlike the cylindrical TMV, CCMV is a "spherical" virus; more specifically, its capsid is a truncated icosahedron consisting of 180 copies of a single gene product, organized as 12 pentamers and 20 hexamers (i.e., the same structure as a soccer ball, or "bucky" [C60] ball). Furthermore, it has been shown that if the RNA of CCMV is replaced by that of another single-stranded (ss) RNA virus (e.g., TMV) - or even by a synthetic anionic polymer - the polymer will be packaged by CCMV capsid protein and virus-like particles will form. These results suggest that there are simple, general, physical principles governing the spontaneous formation ("self-assembly") of viruses from their cationic capsid proteins and anionic genomes. In particular, we are interested in the relationship between the size (i.e., radius of gyration) of ssRNA genomes and the size (diameter) of the protein capsid in which they are spontaneously packaged.
It turns out that very little is known about the radius of gyration of long molecules (1000s of nucleotides) of ssRNA like the genomes of CCMV and TMV. On the experimental side, this is because essentially none of the usual (e.g., small-angle X-ray and neutron) scattering experiments have been done to measure the size and shape of viral genomes. On the theoretical side it is because ssRNA is not a linear polymer, but is characterized instead by branching and long-range interactions - "secondary" and "tertiary" structure resulting from AU/GC pairings of nucleotides from distant portions of its sequence. Accordingly we have begun experiments at both at the Stanford synchrotron X-ray beam line and the neutron scattering facility at NIST, and are at the same time attempting to formulate theoretical models that relate secondary and tertiary structure of viral RNA to its overall size and shape. In addition, we are focusing directly on the packaging of simple, linear, anionic polymers by CCMV capsid protein, in order to clarify the relative importance of polymer size and protein aggregation in determining the size of virus-like particles. Using a polymer like poly(styrenesulphonate) (PSS), for example, we can take advantage of a great deal of quantitative information that is already available for the size and shape (radii of gyration) of the polymer as a function of molecular weight and ionic strength.
The electron micrograph on the right shows the virus-like particles that result from mixing CCMV capsid protein with 3,500,000 MW PSS in "physiological" - "assembly" - buffer, i.e., pH around 7 and salt concentration around 100mM. We find similar results for PSS with a MW of 600,000, but the particles are observed to be somewhat smaller. If no PSS or RNA is added, no capsids of any size are observed under these pH and ionic strength conditions. Note that if the capsids have icosahedral symmetry, i.e., if their structures correspond to one of the Casper-Klug "T" numbers (T=1, 3, 4, 7, 9,...), then the diameters of successively larger particles must increase as sqrt of T. This is only one of the many features we aim to explore, as part of our general investigation of what determines the size of an infectious viral particle.
If one lowers the pH and raises the ionic strength, it is possible to observe the spontaneous self-assembly of a wide variety of structures from purified capsid protein alone, i.e., without the presence of RNA or any other flexible, anionic, polymer. For example, at pH 3.9 in 200mM sodium succinate buffer, we see the formation of monodisperse empty capsids, with size and symmetry corresponding to those of the native, wild-type CCMV virion; see EM picture below on the left. On the other hand, switching to a 100mM sodium cacodylate buffer at pH 5.9, one sees empty (closed-end) cylindrical tubes whose diameters (28 nm) are the same as those of the wild type virions and whose lengths are hundreds of nms long. Mapping out the "phase diagram" of a viral capsid protein is not only important for understanding the sizes and shapes of infectious viruses, but also for the rational syntheses of "nanocontainers" for materials science and biolabel/drug delivery applications.
