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1. US20050276818 - Uncharacterized ORF3 in SARS-coronavirus is a cyclic-AMP-dependent kinase and a target for SARS therapy

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Claims

1. A method for screening for a potential novel antiviral agent effective to inhibit the activity of a viral protein having cyclic-AMP dependent protein kinase activity, comprising:
incubating a mixture containing the viral protein, and an agent to be tested,
measuring cyclic-AMP dependent protein kinase activity,
comparing to cyclic-AMP dependent protein kinase activity in the absence of the agent to be tested, and identifying a potential antiviral agent by the inhibition of cyclic-AMP dependent protein kinase activity in the presence of a test agent.
2. The method of claim 1 in which the antiviral agent inhibits SARS- coronavirus infectivity of a cell.
3. The method of claim 1 wherein the viral protein is cyclic-AMP dependent protein kinase.
4. A method for screening for a potential antiviral agent effective in inhibiting direct interaction of a cyclic-AMP dependent protein kinase and a Calcium-dependent targeting module, comprising:
incubating a mixture containing the cyclic-AMP dependent protein kinase, Calcium-dependent targeting module and an agent to be tested, and
measuring the direct interaction of the cyclic-AMP dependent protein kinase and the Calcium-dependent targeting module,
comparing the degree of direct interaction in the absence of the agent to be tested, and identifying a potential antiviral agent by the inhibition of the binding of a cyclic-AMP dependent protein kinase and a Calcium-dependent targeting module in the presence of a test agent.
5. The method of claim 4 wherein the cyclic-AMP dependent protein kinase is ORF3a from a SARS- coronavirus.
6. The method of claim 4 wherein the Calcium-dependent targeting module is ORF3b from a SARS- coronavirus.
7. The method of claim 4 wherein the degree of direct interaction is measured using GRAMM Hydrophobic Docking Software.
8. The method of claim 4 in which the antiviral agent inhibits SARS- coronavirus infectivity of a cell.
9. A method for screening for a potential novel antiviral agent effective to inhibit the activity of a viral protein having cyclic-AMP dependent protein kinase activity, comprising,
expressing the cyclic-AMP dependent protein kinase protein and the calcium dependent targeting molecule in a yeast cell genetically engineered to increase expression of a reporter gene in the presence of activated PKR protein kinase, and further comprising measuring the level of expression of the reporter gene in the presence and absence of the agent to be tested.
10. The method of claim 9, wherein the reporter gene product is fused to beta.-gal protein.
11. A yeast cell for use in a screen for antiviral agents, which is genetically engineered to express:
(a) a cyclic-AMP dependent protein kinase, and
(b) a calcium dependent targeting module, and
(c) a reporter gene whose expression is increased in response to activation of the PKR protein kinase.
12. The yeast cell of claim 11 wherein the polypeptide containing an ISDR region is NS5A.
13. The yeast cell of claim 12 wherein the reporter gene is a fused .beta.-gal gene.
14. A method of inhibiting SARS- coronavirus infectivity of a cell, comprising administering an effective amount of an agent which interferes with the cyclic-AMP dependent protein kinase activity of said SARS- coronavirus, said agent comprising an antisense molecule complementary to at least one of the ORF3 genes or transcripts.
15. A method for screening for a novel agent effective to inhibit the development of severe acute respiratory syndrome associated with a SARS- coronavirus viral infection, whereby said viral infection is caused by a virus which contains a viral protein with cyclic-AMP protein kinase activity, comprising:
incubating a mixture containing the viral protein, and an agent to be tested, and
measuring cyclic-AMP dependent protein kinase activity,
comparing to cyclic-AMP dependent protein kinase activity in the absence of the agent to be tested, and identifying a potential antiviral agent by the inhibition of cyclic-AMP dependent protein kinase activity in the presence of a test agent.
16. The method of claim 15 wherein the agent inhibits SARS- coronavirus infectivity of a cell line.
17. The method of claim 15 wherein the viral protein is cyclic-AMP dependent protein kinase.
18. A method for screening for an agent effective to inhibit the development of severe acute respiratory syndrome associated with a viral infection, whereby said viral infection is caused by a coronavirus which contains a first viral protein having cyclic-AMP dependent protein kinase activity, whereby said agent is effective in inhibiting the direct interaction of said first viral protein having cyclic-AMP dependent protein kinase activity with a second viral protein being a calcium-dependent targeting module, comprising:
incubating a mixture containing the cyclic-AMP dependent protein kinase, Calcium-dependent targeting module and an agent to be tested, and
measuring the direct interaction of the cyclic-AMP dependent protein kinase and the Calcium-dependent targeting module,
comparing the degree of direct interaction in the absence of the agent to be tested, and identifying a potential antiviral agent by the inhibition of the binding of a cyclic-AMP dependent protein kinase and a Calcium-dependent targeting module in the presence of a test agent.
19. The method of claim 18 wherein the first viral protein is a cyclic-AMP dependent protein kinase.
20. The method of claim 18 wherein the degree of direct interaction is measured using GRAMM Hydrophobic Docking Software.
21. The method of claim 18 in which the antiviral agent inhibits SARS- coronavirus infectivity of a cell.
22. A composition of matter useful for assays involving ORF3 of the coronavirus wherein said composition of matter is selected from the group consisting of; antibodies, sense nucleic acids, antisense nucleic acids, double stranded nucleic acids, target substrates, host cells for recombinant proteins and nucleic acid vectors.
23. A diagnostic screening method to determine the presence of in vivo characteristics of a virus leading to severe acute respiratory syndrome in a mammal comprising the steps of:
a. isolating an in vivo component from a coronavirus of a person to be screened for a characteristic leading to severe acute respiratory syndrome;
b. performing a screening assay using said isolated in vivo component;
c. comparing a result derived from the screening assay with a known value for that characteristic;
d. correlating said in vivo characteristic to a known characteristic leading to severe acute respiratory syndrome, such that the presence of at least one characteristic indicates the virus' level of infectivity.
24. The diagnostic screening method of claim 23, wherein the in vivo component is nucleic acid.
25. The diagnostic screening method of claim 24, wherein the in vivo component is viral RNA.
26. The diagnostic screening method of claim 24, wherein the in vivo component is ORF3.
27. The diagnostic screening method of claim 24, wherein the in vivo component is ORF3a.
28. The diagnostic screening method of claim 24, wherein the in vivo component is ORF3b.
29. The diagnostic screening method of claim 24, wherein the in vivo component is a viral protein kinase.
30. The diagnostic screening method of claim 23, wherein the in vivo component is a protein.
31. The diagnostic screening method of claim 30, wherein the in vivo component is a protein kinase.
32. The diagnostic screening method of claim 31, wherein the in vivo component is a cyclic-AMP dependent protein kinase.
33. The diagnostic screening method of claim 30, wherein the in vivo component is a calcium dependent membrane targeting module.
34. The diagnostic screening method of claim 23, wherein the in vivo component is one or more amino acids included in the sequence of ORF3 protein products.
35. The diagnostic screening method of claim 34, wherein the in vivo component is one or more amino acids included in the sequence of the ORF3a protein product.
36. The diagnostic screening method of claim 34, wherein the in vivo component is one or more amino acids included in the sequence of the ORF3b protein product.
37. The diagnostic screening method of claim 23, wherein the in vivo component is one or more nucleic acids included in the sequence of ORF3 protein products.
38. The diagnostic screening method of claim 37, wherein the in vivo component is one or more nucleic acids included in the sequence of the ORF3a protein product.
39. The diagnostic screening method of claim 37, wherein the in vivo component is one or more nucleic acids included in the sequence of the ORF3b protein product.
40. The diagnostic screening method of claim 23, wherein the screening assay is a genotyping assay.
41. The diagnostic screening method of claim 40, wherein the genotyping assay is an assay selected from the group consisting of restriction fragment length polymorphism, DNA sequencing and RNA sequencing.
42. The diagnostic screening method of claim 41, wherein the genotyping assay determines the nucleic acid sequence of ORF3.
43. The diagnostic screening method of claim 23, wherein the screening assay is a phenotyping assay.
44. The diagnostic screening method of claim 43, wherein the phenotyping assay is an assay selected from the group consisting of protein sequencing, signal transduction cascades, binding assays, western blots and pull-down assay.
45. The diagnostic screening method of claim 44, wherein the phenotyping assay determines the amino acid residue of the ORF3 protein products.
46. A method of screening for agents that modulate the ORF3 protein products of SARS- coronavirus, comprising the steps of:
a. providing a system further comprising;
(i) a peptide similar to the ORF3a protein product that can specifically interact with a peptide similar to ORF3b protein product;
(ii) at least one peptide similar to ORF3b protein product; and
(iii) a reporter system to report the activity of the ORF3 protein products;
b. introducing a test compound to the system; and
c. determining the effect that the test compound has on the system.
47. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the system provided is a system selected from the group consisting of cell lines, recombinant cell lines, extracted proteins, affinity columns and multi-well plates.
48. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the peptide similar to ORF3a protein product is a protein kinase.
49. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the peptide similar to ORF3a protein product is a cyclic-AMP dependent protein kinase.
50. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the peptide similar to ORF3b protein product is a calcium dependent membrane targeting module.
51. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the peptide similar to ORF3a protein product is a fragment at least comprising protein kinase activity.
52. The method of screening for agents that modulate ORF3 activity of claim 51, wherein the peptide similar to ORF3a protein product is a fragment at least comprising cyclic-AMP dependent protein kinase activity.
53. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the peptide similar to ORF3a protein product is a fragment at least comprising protein kinase activity and a surface for interaction with the ORF3b protein product.
54. The method of screening for agents that modulate ORF3 activity of claim 53, wherein the peptide similar to ORF3a protein product is a fragment at least comprising cyclic-AMP dependent protein kinase activity and a surface for interaction with the ORF3b protein product.
55. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the peptide similar to ORF3b protein product is a fragment at least comprising calcium dependent membrane trafficking module activity.
56. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the peptide similar to ORF3b protein product is a fragment at least comprising calcium dependent membrane trafficking module activity and a surface for interaction with the ORF3a protein product.
57. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the ORF3 protein products comprise at least one polymorphism.
58. The method of screening for agents that modulate ORF3 activity of claim 46, wherein ORF3 nucleic acid comprises at least one polymorphism.
59. The method of screening for agents that modulate ORF3 activity of claim 46, wherein at least one of the ORF3 protein products has a shifted amino acid sequence configuration caused by sequence variations selected from the group consisting of splice variants, truncations, additions and fusion proteins.
60. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the reporter system is a system selected from the group consisting of two-stage antibody detection, electrophoresis, reporter construct vectors, ELISA, affinity columns, plaque assays and enzyme substrate phosphorylation assays.
61. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the test compound is selected from the group consisting of a peptide, polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecule, a small inorganic molecule, and a nucleotide sequence.
62. The method of screening for agents that modulate ORF3 activity of claim 46, wherein the step of determining the effect a test compound has on the system includes comparison of the system in the presence of the test compound with the system in the absence of the test compound.
63. A modulator of ORF3 mediated viral infectivity identified using the method of claim 46.
64. A method for the treatment of severe acute respiratory syndrome comprising the steps of administering to a patient diagnosed with having SARS-CoV an agent that modulates the consequences of the ORF3 protein products on the infectivity rate for said SARS-CoV in a quantity sufficient to reduce said rate of infectivity.
65. The treatment method of claim 64, wherein the agent that modulates the consequences of the ORF3 protein products on the infectivity rate for said SARS-CoV is an agent selected from the group consisting of a peptide, polypeptide, peptidomimetic, non-peptidyl compound, carbohydrate, lipid, a synthetic compound, a natural product, an antibody or antibody fragment, a small organic molecule, a small inorganic molecule, and a nucleotide sequence.
66. An isolated nucleic acid molecule including the ORF3 open reading frame.
67. An isolated nucleic acid molecule including the ORF3a open reading frame.
68. An isolated nucleic acid molecule including the ORF3b open reading frame.
69. An isolated nucleic acid molecule that encodes a polypeptide sequence identical to the polypeptide sequence encoded by the ORF3 open reading frame.
70. An isolated nucleic acid molecule that encodes a polypeptide sequence identical to the polypeptide sequence encoded by the ORF3a open reading frame.
71. An isolated nucleic acid molecule that encodes a polypeptide sequence identical to the polypeptide sequence encoded by the ORF3b open reading frame.
72. An isolated nucleic acid molecule that encodes a polypeptide sequence that varies from the polypeptide sequence encoded by the ORF3 open reading frame by one or more conservative amino acid substitutions.
73. The isolated nucleic acid molecule of claim 72 in which there is from 1 to 20 conservative amino acid substitutions.
74. The isolated nucleic acid molecule of claim 73 in which there is from 1 to 10 conservative amino acid substitutions.
75. An isolated nucleic acid molecule that encodes a polypeptide sequence that varies from the polypeptide sequence encoded by the ORF3a open reading frame by one or more conservative amino acid substitutions.
76. The isolated nucleic acid molecule of claim 75 in which there is from 1 to 20 conservative amino acid substitutions.
77. The isolated nucleic acid molecule of claim 76 in which there is from 1 to 10 conservative amino acid substitutions.
78. An isolated nucleic acid molecule that encodes a polypeptide sequence that varies from the polypeptide sequence encoded by the ORF3b open reading frame by one or more conservative amino acid substitutions.
79. The isolated nucleic acid molecule of claim 78 in which there is from 1 to 20 conservative amino acid substitutions.
80. The isolated nucleic acid molecule of claim 79 in which there is from 1 to 10 conservative amino acid substitutions.