Methods and compositions for the identification of anti-poxvirus agents
View Patent ↗The invention provides methods for identifying an anti-poxviral agents. In many embodiments, the methods involve contacting a poxviral p28 polypeptide with a candidate agent, and determining an effect of the agent on a ubiquitin ligase activity of the p28 polypeptide. The effect of the agent may be determined using a variety of different cell based or biochemical assays, such as polyubiquitylation assays and cell viability assays. The invention also provides methods for modulating poxvirus pathogenicity in a cell, and methods of treating an individual infected with a poxvirus. The subject methods find use in a variety of drug discovery, research and military applications.
1. A method of ubiquitylating a substrate, comprising:
combining an E1 polypeptide, an E2 polypeptide, ubiquitin and a poxvirus P28 protein having an amino acid that is at least 80% identical to a p28 protein encoded by the genome of an orthopoxvirus under ubiquitylation reaction conditions; and
detecting a ubiquitylated substrate.
2. The method of claim 1 , wherein said ubiquitylated substrate is autoubiquitylated p28 polypeptide.
3. The method of claim 1 , wherein said ubiquitylated substrate is a polyubiquitinated substrate.
4. The method of claim 1 , wherein said ubiquitylated substrate is a cognate poxviral polypeptide or a host-cell polypeptide.
5. The method of claim 1 , wherein said method is conducted in a cell as a cell-based method.
6. The method of claim 1 , wherein said method is a cell-free ubiquitylation method.
7. The method claim 1 , wherein said method is performed in the presence of a test agent.
8. The method of claim 1 , wherein said poxvirus P28 protein has an amino acid that is at least 95% identical to a p28 protein encoded by a variola virus genome.
9. The method of claim 1 , wherein said poxvirus P28 protein has an amino acid that is identical to a p28 protein encoded by a variola virus genome.
10. A method for identifying an inhibitor of poxvirus p28 polypeptide ubiquitin ligase activity, comprising:
contacting a poxvirus p28 polypeptide having an amino acid that is at least 80% identical to a p28 protein encoded by the genome of an orthopoxvirus with a candidate agent in the presence of ubiquitin under ubiquitylation reaction conditions; and
determining an effect of said agent on a ubiquitin ligase activity of said p28 polypeptide;
wherein the effect of the candidate agent upon said ubiquitin ligase activity is indicative of its inhibitory effect.
11. The method of claim 10 , wherein said activity is a polyubiquitylation activity.
12. The method of claim 10 , wherein said activity is an autoubiquitylation activity.
13. The method of claim 10 , wherein said method further comprises:
contacting said candidate agent with cells comprising said poxvirus p28 polypeptide and ubiquitin; and
determining an effect of said agent on said cells.
14. The method of claim 13 , wherein said cells are tested for p28-induced cell death.
15. The method of claim 13 , wherein said cells are tested for UV-induced apoptosis.
16. The method of claim 13 , wherein the said cells are tested for p28-mediated antagonism of cytokine activation.
17. The method of claim 16 , wherein said cyctokines include a tumor necrosis factor and an interferon.
18. The method of clam 10 , wherein said method further comprises contacting said candidate agent with a poxvirus-infected cell.
19. The method of clam 10 , wherein said method further comprises testing said candidate agent on a poxvirus-infected mammal.
20. The method of clam 19 , wherein said mammal is a non-human animal model of poxvirus infection.
21. The method of claim 10 , wherein said method includes combining an E1 polypeptide, an E2 polypeptide, ubiquitin and a poxvirus P28 protein under ubiquitylation reaction conditions in the presence of a test agent.
22. The method of claim 21 , wherein said E1 polypeptide is human ubiquitin E1.
23. The method of claim 21 , wherein said E2 polypeptide is Ubc4, UbcH5 or Ubc13.
24. The method of claim 10 , wherein said method is a cell-based method.
25. The method of claim 10 , wherein said method is a cell-free method.
26. The method of claim 10 , wherein said poxvirus P28 protein has an amino acid that is at least 95% identical to a p28 protein encoded by a variola virus genome.
27. The method of claim 10 , wherein said poxvirus P28 protein has an amino acid that is identical to a p28 protein encoded by a variola virus genome.
28. A method for identifying a target for the treatment of a poxvirus infection, comprising,
contacting a poxvirus p28 polypeptide having an amino acid that is at least 80% identical to a p28 protein encoded by the genome of an orthopoxvirus with a candidate cellular polypeptide in the presence of ubiquitin and under condition suitable for p28 ubiquitylation activity; and,
determining if said polypeptide is ubiquitylated by said p28 polypeptide;
wherein a cellular polypeptide that is ubiquitylated by said p28 polypeptide is a target for modulation to effect treatment of a poxvirus infection in a cell.
29. The method of claim 28 , wherein said method is a cell-based method.
30. The method of claim 28 , wherein said method is an in vitro polyubiquitylation assay.
31. The method of claim 28 , wherein said poxvirus P28 protein has an amino acid that is at least 95% identical to a p28 protein encoded by a variola virus genome.
32. The method of claim 28 , wherein said poxvirus P28 protein has an amino acid that is identical to a p28 protein encoded by a variola virus genome.