Screening method for orthopoxvirus antivirals
View Patent ↗The focus of the present invention is an effective anti-poxvirus drug for use in treating or preventing human disease caused by pathogenic poxviruses. More particularly, the present invention relates to antiviral drugs that target the poxvirus proteinase responsible for core protein maturation, a step which is absolutely essential for the production and spread of infectious virions.
1. An assay comprising the steps of:
infecting cultured cells with vaccinia virus;
co-transfecting the cells with one or more plasmids comprising DNA encoding the I7L protease enzyme and at least one substrate, such that both the substrate protein and enzyme are constitutively expressed; and
measuring the amount of cleavage product of the I7L protease.
2. The assay of claim 1 , wherein the cultured cells are infected with temperature-sensitive vaccinia virus at a non-permissive temperature.
3. The assay of claim 1 , wherein the cultured cells are infected with wild-type vaccinia virus at a permissive temperature.
4. The assay of claim 1 , wherein the substrate protein and enzyme are expressed from a synthetic early-late promoter.
5. The assay of claim 1 , wherein the substrate protein is a core protein.
6. The assay of claim 5 , wherein each core protein comprises a tag on the C-terminus of the protein.
7. The assay of claim 6 , wherein the tag is a FLAG epitope.
8. The assay of claim 1 , wherein the substrate is P25K also known as L4R.
9. The assay of claim 1 , wherein the substrate is P4a also known as A10L.
10. The assay of claim 1 , wherein the substrate is P4b also known as A3L.
11. The assay of claim 1 , wherein the substrate is AI7L.
12. The assay of claim 1 , wherein the vaccinia virus has a ts mutation in the I7L gene.
13. The assay of claim 1 , wherein the cultured cells are BSC 40 cells.