We are a group of physical chemists, biophysicists and molecular biologists, trying to figure out how viruses “work” from a physical-science point of view, and how to use reconstituted non-infectious forms of them – virus-like particles – for biotechnology and translational medicine purposes.
Bill and Chuck started the lab in 2003, after each had worked for decades (!) in fields that have little (actually, nothing) to do with viruses (or anything else biological, for that matter). Bill had carried out theoretical work on the statistical mechanics and optical properties of simple liquids and on phase transitions in liquid crystals and self-assembling systems; and Chuck had done experimental work on critical phenomena in liquid solutions, kinetics of phase transitions, and the physics of molecular momolayers on liquid and solid surfaces.
Current projects involving the physical aspects of viruses:
- 3D Cryo-Electron Microscopy (CryoEM) Reconstructions of RNA Inside Viral Capsids
- Distribution of RNA Ends in Multiplet-Capsid Structures
- In vitro Synthesis of Enveloped Virus-Like Particles (EVLPs)
- RNA Homopolymer Packaging by CCMV - the Role of RNA Secondary Structure on Self-Assembly
- Catching the Ends of Fluctuating RNA Outside Intact Capsids
- Characterization of Assembly Mechanism through Cryo-Electron Tomography (CryoET) of Individual RNA-Capsid Protein Assembly Intermediates
- CryoET Reconstructions of B1, B2, and B3/4 Virions
- Atomistic Molecular Dynamics Studies of Protein-RNA Interactions
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 experiments (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 simplest 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 are organized into groups of 12 pentamers and 20 hexamers, involving 3 sets of inequivalent positions.
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 capsid proteins 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 capsid protein 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, and also to prepare well-characterized and effective 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 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 that contain genes of interest in (single-stranded) RNA form. The RNA form is essential for enabling spontaneous self-assembly of the gene into a virus-like particle when mixed with the right viral capsid protein; this wouldn't be possible with (double-stranded) DNA. For self-assembly purposes, the "right" capsid protein 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 capsid protein 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 virus-like particles 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 above 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") to the open reading frame (ORF) coding for the RNA-dependent RNA polymerase. 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 a million copies of the original molecule. "T2A" encodes a self-cleaving peptide sequence that ensures independence of the RNA replicase and the gene product of the GOI.
Current projects involving the delivery of packaged, self-amplifying, RNA genes:
- Design and synthesis of self-replicating forms of mRNA (replicons)
- Delivery of microRNA replicons as a pancreatic cancer treatment
- In vitro reconstituted VLPs for delivery of microRNA replicons as a pancreatic cancer treatment
- In vitro reconstituted CCMV VLPs for delivery of self-amplifying cancer antigens
- In vitro reconstituted TMV VLPs for delivery of chemo and Sindbis RNA therapeutics as an ovarian cancer treatment
- Delivery of self-amplifying adjuvants to trigger interferon production
- Defective interfering RNA as a Yellow Fever anti-viral
- Targeted delivery of gene therapies through protein fusions and chemical conjugations of ligands to reconstituted VLPs
Our first experiment on viruses (PNAS 2003), measuring directly the pressure in DNA bacterophages, grew out of theoretical work on DNA condensation and packaging in viral capsids (see PNAS 2001 and Biophys. J. 2003) . Earlier, during Bill's 1999 sabbatical stay in Paris, discussions with groups at the Curie Institute and the University of Paris-Sud had resulted in cryo-electron-microscopy imaging of these phenomena.
The cryo-EM picture above (PNAS 2000) shows three bacterial viruses -- T5 particles, colored green -- that have been fooled into thinking they are infecting a bacterial cell. What we did was to prepare phospholipid vesicles ("liposomes") reconstituted with several copies of the receptor protein that is normally on the outer membrane of the bacterial host for T5. When the tip of the tail of the virus binds the receptor, ejection of the (pressurized) DNA genome is triggered. But instead of entering a bacterial cytoplasm where copies of the virus are made and its genes expressed, each genome enters the aqueous interior of a liposome. And because of polyvalent cations that we have put inside the liposomes, the three DNA molecules are condensed into a hexagonally-packed toroid (blue); the circumferential winding of the individual DNA strands (duplexes) are clearly visible.The left-most virus hasn't yet bound a receptor, so its DNA is still confined in its "head" (red).