In collaboration with Gary Suizdak at Scripps, we have been using mass spectroscopy to examine the dynamics of the human rhinovirus capsid. MALDI (matrix assisted laser desorption and ionization) is a mass spectroscopy method that can, with great precision, determine the molecular weights of biological specimens. As described in the figure below, the specimen is mixed with the matrix material and then ionized with a laser. The longer it takes for the ionized species to travel to the detector, the larger its molecular weight.
In the above experiment we obtained a rather unexpected result. When intact rhinovirus is icubated with trypsin, cleavage occurs in a very short period of time. The unusual part is the fact that this cleavage is occuring at internal sites that are not accessible to solvent. Shown above in the structure of HRV14 (VP1=blue, VP2=green, VP3=red, and VP4=mauve). The RNA interior is towards the bottom of the figure. The trypsin cleavage sites are denoted by yellow balls. Note that VP4, which is completely buried by the capsid, is the most sensitive to cleavage. Since matrix-bound trypsin causes the same effect, we know that this digestion is not due to penetration of the enzyme into the capsid interior. This leaves only two explanations for these results; some of the capsids are denatured and being digested or the capsid is 'breathing' and transiently exposing the termini to the exterior. To determine which was the correct model, we used the antiviral WIN compounds.
In the figure above, the WIN drug is represented by a space-filling model. We know that WIN compounds stabilize the virion against heat and acid denaturation. Therefore, we suspected that the drugs would also control the 'breathing'. When WIN drugs were added to the digestion mixture, no trypsin cleavage occurred. This clearly demonstrated that the trypsin cleavage in the first experiment was not due to 'dead' particles and also shows that WIN drugs block 'breathing'. We believe that this 'breathing' is part of the normal infection process that is agonized by ICAM binding (the receptor).
	As a final control, we also added WIN compounds to another virus/trypsin reaction. In this experiment, WIN compounds were found to not block digestion of another virus (Flock House). Since we knew that WIN drugs neither inhibited or bound to the virus, this served to prove that the WIN inhibition of digestion is not due to the drug attacking the enzyme itself.
Finally, to show these results in a different way, the cleavage sites are mapped onto a portion of the virion surface (white balls). The figure on the left is a surface rendering of the HRV14 capsid and color coded according to the surface features- the deep depressions are in blue and the protrusions are red. It is interesting to note that there is not only a depression about the 5-fold axes, but there is also a depression at the 2-fold axis. Furthermore, these cleavage sites seem to cluster near the 2-fold axes (center of the figure on the right). The capsid is very thin at this point and it may be that the 'breathing' opens up this area and allows the termini to be extruded. The WIN compounds bind and block these conformational changes as does lowering the temperature of the particle. On the other hand, the receptor probably increases these dynamic fluctuations and allows the RNA to be injected into the target cell.

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