Research

Virus particles can be as simple as a molecule of RNA or DNA inside a spherical shell - the capsid - made up of multiple copies of a single protein. Further, because viruses only "become alive" when they are inside their hosts, it is possible to study them -- outside their hosts, in a test tube -- as physical objects, i.e., to do the same controlled experiment (and theory) on them that one routinely does with more familiar polymer and colloidal systems.

This approach is best illustrated with the example of one of the simple viruses we work on - cowpea chlorotic mottle virus (CCMV) - a plant virus with a single-stranded RNA genome. Each particle of CCMV is a one-molecule-thick spherical shell consisting of (exactly!) 180 copies of a single protein, surrounding and protecting single copies of RNA genes of the virus. The 180 capsid proteins (CPs) are organized into groups of 12 pentamers and 20 hexamers, involving 3 sets of inequivalent positions (corresponding to a Caspar-Klug triangulation number of T=3), labeled in blue ("A"), red ("B"), and green ("C") in the figure to the right.

Most remarkably, this icosahedrally-symmetric (soccer-ball-like/"Bucky-ball"-like) structure can form spontaneously: one simply has to mix the purified RNA molecules and purified CPs under the right conditions of pH and ionic strength, and a virus forms that is infectious and indistinguishable from those found in infected plants. By changing these solution conditions, and replacing the viral RNA by non-viral RNA (and even by synthetic anionic polymers) and replacing the wildtype CP by genetically- and/or chemically- modified forms of it, we are able to learn a lot about the fundamental physical chemistry of this biological process - the formation of an infectious virus. Similarly, we can prepare well-characterized virus-like particles (VLPs) that contain therapeutic RNA instead of viral RNA and that can be conjugated with antibodies and other protein ligands to provide powerful gene delivery systems.

As a result of billions of years of evolution, what viruses do better than anything else - better than anything else they do, or any thing else does - is to protect and deliver genes to specific (targeted) cells. This is why it is impossible not to consider their use as a gene delivery systems for medical purposes. To avoid the complications and potential dangers of using "attenuated" or "inactivated" viruses, however, we pursue a research program that involves the design and synthesis - from purified components in test tubes - of virus-like particles (VLPs) that contain genes of interest in single-stranded RNA (ssRNA) form. The ssRNA form is essential for enabling spontaneously self-assembly of the gene into a VLP when mixed with the right viral capsid protein (CP); this isn't possible, for example, with double-stranded DNA (dsDNA). For self-assembly purposes, the "right" CP is one that will efficiently package any RNA, independent of its sequence, as long as it isn't too long or too short. We have found that CP from cowpea chlorotic mottle virus (CCMV) or brome mosaic virus (BMV) does this job. The other advantage of RNA genes is that it is possible to work with them in messenger-sense and self-amplifying form, enabling them to be strongly replicated in the cytoplasm of targeted host cells before being translated into proteins of interest.

The scenario outlined above - for in vitro reconstituted VLPs consisting of self-amplifying RNA inside a protective protein shell - mimics closely the life cycle of many of the simplest and most common viruses. The figure to the left shows the replication and protein synthesis scheme for molecule 1 of the two-molecule RNA genome of the insect virus Nodamura, into which we have added a gene of interest (GOI) at the end of the open reading frame (ORF) coding for the RNA-depended RNA polymerase (RdRp). This RNA replicase protein binds the RNA molecule (that encodes it and the GOI) and generates large numbers of reverse-complement (negative-sense) strands that are in turn transcribed by the replicase to make up to hundreds of thousands of copies of the original molecule. T2A encodes a self-cleaving peptide sequence that ensures the independence/function of the RNA replicase and gene product of the GOI.