Previous Work

How we got started thinking about viruses....

Three bacteriophages trying to infect an empty liposome. The tips of the tails of these three T5 viruses (green) have bound to receptor proteins that have been reconstituted in liposomes and, driven by the high pressure in their capsids, the phage have ejected their DNA genomes. These DNA molecules are then condensed into hexagonally-packed toroid inside the liposomes, by the high concentration of polyvalent cations there.

Our first experiment on viruses [1], measuring directly the pressure in DNA bacterial viruses (“phages”), grew out of theoretical work on DNA condensation and packaging in viral capsids[2, 3]. Earlier, during Bill's 1999 sabbatical stay in Paris, discussions with groups at the Curie Institute and the University of Paris-Sud had led to the design of a series of in vitro studies of genome ejection from DNA viruses.

To the left is a cryo-EM image showing three bacterial viruses – T5 particles, colored green – that have been fooled into thinking they are infecting a bacterial cell. What we did [4] was to prepare phospholipid vesicles ("liposomes") that were 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 its genes are expressed and copies of the virus are made, each genome enters the empty 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) is clearly visible. The left-most virus hasn't yet bound a receptor, so its DNA is still confined in its "head" (red).

The figure to the right shows how we measured the pressure of the lambda bacteriophage genome in its capsid. This pressure, which we predicted to be as high as tens of atmospheres [2, 3] is due to the fact that the highly-charged DNA molecule is packed at essentially close-packed density, giving rise to a large self-repulsion energy. Also, because the persistence length of DNA is significantly smaller than the radius of the capsid inside which it is confined, it is strongly bent all along its length, giving rise to a large elastic energy. Accordingly, a great deal of work needs to be done to package the genome into the preformed capsid, and this stored energy-per-unit volume – pressure! – is then available to drive ejection when opening of the capsid is triggered by its binding of host cell receptor.

The top cartoon in the figure shows a solution of purified phage lambda (the red-lollipop-looking things) to which have been added the well-calibrated osmolyte PEG8000 (green) and some DNaseI (not shown). Upon addition of the membrane protein receptor (solubilized by detergent), genome ejection is initiated and proceeds until the pressure associated with the DNA remaining in the capsid has dropped to the value of the osmotic pressure arising from the controlled concentration of PEG8000. Ejected DNA is digested by the DNase and assayed by UV absorbance of the supernatant after centrifugation, and unejected DNA is sedimented with the capsids, extracted, and its length determined in an electrophoretic gel assay. In this way we determine the pressure in the capsid as a function of the amount of DNA inside.

Measuring the pressure in DNA virus. Purified lambda phage are incubated in a solution with osmotic pressure set by PEG8000. After adding the receptor that triggers genome release, ejection occurs until the pressure in the capsid has dropped to the osmotic pressure; the amount of ejected DNA then tells us the capsid pressure as a function of remaining DNA length.

[1] Evilevitch, A., Lavelle, L., Knobler, C. M., Raspaud, E., & Gelbart, W. M. (2003). Osmotic pressure inhibition of DNA ejection from phage. Proceedings of the National Academy of Sciences, 100, 9292-9295.

[2] Kindt, J., Tzlil, S., Ben-Shaul, A., & Gelbart, W. M. (2001). DNA packaging and ejection forces in bacteriophage. Proceedings of the National Academy of Sciences, 98, 13671-13674.

[3] Tzlil, S., Kindt, J. T., Gelbart, W. M., & Ben-Shaul, A. (2003). Forces and pressures in DNA packaging and release from viral capsids. Biophysical journal, 84, 1616-1627.

[4] Lambert, O., Letellier, L., Gelbart, W. M., & Rigaud, J.-L. (2000). DNA delivery by phage as a strategy for encapsulating toroidal condensates of arbitrary size into liposomes. Proceedings of the National Academy of Sciences, 97, 7248-7253.