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Research Overview

What are the Physical Properties - Branching Characteristics and 3D sizes - of viral-length RNA molecules?

The figure to the left is a cryo-electron microscopy image that we obtained [RNA 2012] from a purified solution of the RNA molecules (circled in red) corresponding to one of the genes of CCMV. Each of the molecules in the micrograph is chemically identical to the others – the same 3200 nucleotide (nt)-long sequence of RNA. But they appear different because they have different secondary and tertiary structures and because they have different orientations in the vitreous water in which they are trapped at low temperature; accordingly, they have different 2D projections in the transmission micrograph. Indeed, an RNA molecule this long must be regarded as a “statistical object” that must be represented by an ensemble of configurations, much like a long semi-flexible polymer.

How can in vitro packaged self-amplifying RNA genes be used for in situ expression of proteins


In our general introduction of "In Vivo Self Amplifying RNA research projects", we emphasized how important it is to mimic the natural use of RNA replicons by a wide range of positive-strand RNA viruses, for purposes of high-level protein expression. We featured the particular case of Nodamura virus, with its two-molecule genome consisting of RNA1 coding for the RNA replicase (RdRp) and RNA2 coding for the capsid protein. One way to use this system for delivery of genes of interest (GOIs) is to simply insert the GOI into the end of RNA1, immediately following a self-cleaving proteolytic sequence, so that the GOI RNA is replicated along with RNA1 and so that its gene product – the desired therapeutic or reporter protein – will be cleaved in functional form from the RdRp. We have done this using EYFP as the reporter gene.

In Vivo Self-Amplifying RNA Research Projects

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By “in vivo” experiments we mean ones performed in host cells (rather than in host animals, which is how the term “in vivo” is more generally used, in virology and medical contexts). And by “host cells” we mean controlled monolayers of cells in petri dishes. In this classical form the cells can easily be transfected by RNA or VLPs, or infected by virus, and the transformed cells can easily be assayed in a large number of ways. Ultimately, we would like to transform and assay cells that have been targeted in animals, but we need first to demonstrate and understand how changes of interest can be effected under the controlled conditions of cell culture.

IN VITRO/PHYSICAL CHEMISTRY PROJECTS

 

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 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.

Our Groups' Previous Work in Studying the Pressure of DNA Viruses - Packing DNA in Bacteriophage

Bacteriophage lambda, shown in the electron micrograph, consists of a protein capsid 30 nm in radius that has a long cylindrical tail. Its genome, double stranded DNA (dsDNA), is protected by the capsid from attack by nuclease enzymes that would break it down into its nucleotides and therefore lose the genetic information needed to replicate the phage. The DNA contains 48.6 kilo-base pairs; if it were fully extended it would be 17 micrometers long. When the phage is replicated in the host cell, an early form of the capsid, the procapsid, is formed and the DNA is driven into it by a molecular motor at one of the procapsid vertices. This is quite feat! Imagine packing a length of string into an object that is only 1/400th its size. To make the job harder, add negative charges to the string and make it stiff. The stiffness of ds DNA is very high; a measure of this stiffness is its persistence length. It is difficult to bend objects on a scale smaller than the persistence length. The persistence length of dsDNA is 50 nm, and to bend it so that it can fit into the capsid is therefore highly costly in energy.

Some of Our Group's Previous Work - Squeezing Viruses with an Atomic Force Microscope

Pressures on capsids
We know from our studies with lambda phage that viral capsids can support internal pressures of 50 - 60 atm. The interactions between the proteins that make up the capsid are held together by hydrophobic and electrostatic forces and hydrogen bonds. How can structures that are joined by relatively weak bonds be so strong? Just how strong are viral capsids?

The Origin of Icosahedral Symmetry in Viruses

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Certainly one of the most intriguing facts about viruses is that the large majority of them display full icosahedral symmetry, arguably the highest and also the most esthetically-pleasing symmetry shown in Nature. The elements of icosahedral symmetry involve 6 five-fold rotation axes, 10 three-fold, and 15 two-fold. The figure to the right shows a number of examples, including the 60nm-diameter human papilloma virus at one end and 28nm CCMV near the other; similar image reconstructions for still larger viruses, up to the 100nm-diameter herpes simplex virus, are available from cryo-EM and X-ray work (Figure from Review by Baker et al.).
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