Methods of preventing and treating viral infections by inhibiting the deISGylation activity of OTU domain-containing viral proteins
Viruses having an impaired ability to deISGylate ISG15 conjugates, in particular, viral mutants comprising a mutation in the viral genome that reduces or eliminates the ability of the viral OTU domain-containing protein encoded by the viral genome to deISGylate ISG15 conjugates and/or deubiquitinate ubiquitinated proteins and/or deNeddylate Neddylated proteins are disclosed. Such viral mutants may be used in the formulation of immunogenic compositions for inducing an immune response and preventing, managing and/or treating a viral infection. Also disclosed are methods for identifying anti-viral compounds, in particular, methods of identifying compounds that reduce or inhibit the deISGylation activity and/or deubiquitination and/or deNeddylation activity of a viral OTU domain-containing protein. The compounds identified using such methods may be used as antiviral agents for the prevention, treatment and/or management of viral infections.
1. A method for identifying a compound that modulates the deISGylation activity of a viral OTU domain-containing protein or fragment thereof comprising the OTU domain, comprising:
a. contacting a compound with a composition comprising ISG15 conjugated protein and the viral OTU domain-containing protein or fragment thereof, and
b. determining the amount of ISG15 conjugated protein in the composition, wherein an alteration in the amount of ISG15 conjugated protein relative to a negative control indicates that the compound modulates the deISGylation activity of the viral OTU domain-containing protein or fragment thereof.
2. The method of claim 1 , wherein a compound with antiviral activity is identified if there is an increase in the amount of ISG15 conjugated protein.
3. The method of claim 1 , wherein the compound decreases the deISGylation or deubiquitination activity of the viral OTU domain-containing protein.
4. The method of claim 1 , wherein the viral OTU domain-containing protein is a nairovirus OTU domain-containing protein.
5. The method of claim 1 , wherein the viral OTU domain-containing protein is the L protein of a nairovirus.
6. The method of claim 5 , wherein the nairovirus is Crimean Congo Hemorrhagic Fever Virus (CCHFV) or Dugbe virus.
7. The method of claim 1 , wherein the viral OTU domain-containing protein is an arterivirus or herpes virus OTU domain-containing protein.
8. A method for identifying a compound that modulates the deISGylation activity of a viral OTU domain-containing protein, comprising:
a. contacting a compound with a composition comprising an artificial substrate that mimics ISG15 cleavage from a protein to which it is conjugated and the viral OTU domain-containing protein, and
b. measuring the amount of cleavage of the artificial substrate, wherein an alteration in the cleavage of the artificial substrate relative to a negative control indicates that the compound modulates the deISGylation activity of the viral OTU domain-containing protein.
9. A method for identifying a compound that reduces the deISGylation activity of a viral OTU domain-containing protein, comprising:
a. contacting a compound with a composition comprising an artificial substrate that mimics ISG15 cleavage from a protein to which it is conjugated and the viral OTU domain-containing protein, and
b. measuring the amount of cleavage of the artificial substrate, wherein a decrease in the cleavage of the artificial substrate relative to a negative control indicates that the compound reduces the deISGylation activity of the viral OTU domain-containing protein.
10. The method of claim 8 , wherein the artificial substrate is ISG15 fused in its carboxy-terminus to a fluorogenic substrate.
11. The method of claim 9 , wherein the artificial substrate is ISG15 fused in its carboxy-terminus to a fluorogenic substrate.
12. The method of claim 10 , wherein the fluorogenic substrate is 7-amido-4-methylcoumarin (AMC).
13. The method of claim 11 , wherein the fluorogenic substrate is 7-amido-4-methylcoumarin (AMC).
14. The method of claim 8 , wherein the artificial substrate is a synthetic AMC substrate that incorporates the carboxy-terminal six residues found in ISG15 (LRLRGG; SEQ ID NO:1).
15. The method of claim 9 , wherein the artificial substrate is a synthetic AMC substrate that incorporates the carboxy-terminal six residues found in ISG15 (LRLRGG; SEQ ID NO:1).
16. The method of claim 8 , wherein the artificial substrate comprises DABCYL or EDANS moieties on opposite ends of a 12 amino acid peptide which contains the cleavage site LRLRGG (SEQ ID NO:1).
17. The method of claim 9 , wherein the artificial substrate comprises DABCYL or EDANS moieties on opposite ends of a 12 amino acid peptide which contains the cleavage site LRLRGG (SEQ ID NO:1).
18. The method of claim 8 , wherein the viral OTU domain-containing protein is a nairovirus OTU domain-containing protein.
19. The method of claim 8 , wherein the viral OTU domain-containing protein is the L protein of a nairovirus.
20. The method of claim 9 , wherein the viral OTU domain-containing protein is the L protein of a nairovirus.
21. The method of claim 19 , wherein the nairovirus is Crimean Congo Hemorrhagic Fever Virus (CCHFV) or Dugbe virus.
22. The method of claim 20 , wherein the nairovirus is Crimean Congo Hemorrhagic Fever Virus (CCHFV) or Dugbe virus.
23. The method of claim 8 , wherein the viral OTU domain-containing protein is an arterivirus or herpes virus OTU domain-containing protein.
24. The method of claim 9 , wherein the viral OTU domain-containing protein is an arterivirus or herpes virus OTU domain-containing protein.
25. The method of claim 9 , wherein the viral OTU domain-containing protein is a nairovirus OTU domain-containing protein.