Computational Science
Variola virus is the most virulent member of the orthopox genus of viruses. It is specific for humans and has no other animal hosts. A prototype for the orthopox viruses is vaccinia virus, which shows considerable sequence similarity to variola yet does not lead to disease in humans. In the absence of three-dimensional structures for variola proteins, it should be possible to substitute those obtained from the highly similar vaccinia virus.
One such structure is that of the Type I topoisomerase. This class of enzyme plays an important role in DNA replication and transcription and has been proposed as a putative target for anticancer therapy. The principal role of Type I is the relaxation of supercoiled DNA through the cleavage of a single strand of a DNA double helix (see below for more details). Unwinding, followed by recombination, can then take place. While these enzymes are present in other organisms, they are particularly important for compact viral genomes, making them an ideal target for intervention.
The structure of vaccinia topoisomerase contains two domains but it has been shown that the isolated C-terminal domain is catalytically competent. Sequence analysis of topoisomerases has also shown that certain motifs are conserved in the active site: these include an RHRW group that appears to be involved in stabilizing reaction intermediates, and an SKxxY motif that is directly involved in forming the covalent linkage between the protein and DNA. Initially we will screen compounds against the second site, as the first is not completely resolved in the vaccinia crystal structure.
While this is running on the Metaprocessor, Evotec OAI will build homology models of the missing residues, allowing us to predict their positions within the site. The model building will involve the generation of many possible conformations for the missing region of structure. The best of these will then be checked for stability using molecular dynamics simulations before selection of the final model(s). A site-finding tool will be used to predict the most likely area for drug molecules to bind, and this will be taken forward into a screen on the Metaprocessor.
A second issue that will also be addressed is that of specificity for the variola/vaccinia protein over that of the human host. Crystal structures exist for the human type I protein allowing secondary screening to be carried out. Only those molecules that fit the viral protein but not that from humans need be considered further. A second advantage of such an approach is that the inhibitors of the human enzyme might show some promise as agents for cancer therapy. Thus the screening project will identify not just potential agents against smallpox but also putative anti-cancer drugs.
Cell Biology and Virus Biochemistry
The variola virus "smallpox" is a very large "brick-shaped" particle 200-400 nm long (shown of the front page of the web site). The virus is extremely complex, it contains an outer ridged membrane and an independent inner core membrane structure (dumbbell shaped). The inner structure contains the tightly compressed virus nucleoprotein DNA, along with over a 100 proteins and at least 10 viral enzymes. These enzymes are used in nucleic acid metabolism and genome replication. The variola virus DNA genome contains approximately 250, 000 base pairs in the form of about 200 genes. The figure below shows a schematic of the replication of the variola virus.
The replication of the variola virus follows the following steps:
1) Binding to cell receptors and penetration - how this happens is not known and there are probably a range of receptors suitable and a number of different pathways the variola virus enters the cell.
2) Uncoating - this occurs in two stages, first the removal of the outer coat, then when inside the cell, the inner coat is removed to release the variola virus proteins, enzymes and supercoiled DNA sequence.
3) Gene expression and transcription - this is carried out by viral gene expression enzymes and takes place in two phases; i) formation of early genes producing approximately 50% of the genome, the Type I topoisomerase enzymes are involved in this stage to uncoil the DNA. Followed by ii) late gene expression after DNA replication has taken place.
4) Genome replication - this process forms large concatemers which are cleaved to form virus genomes.
5) Assembly and release - occurs in the cell cytoskeleton via complicated processes followed by cell release or even transfer to adjacent cells. Type I topoisomerase enzymes are also involved in the assembly process to suppercoil the DNA ready for release.
The virus replication processes of steps 1-5 is very fast (~12 hrs) to such a large complex virus. The over production of the variola virus often results in host cell death and ultimately 30-40% chance of the host death.
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