Claims
- 1. An isolated protein complex comprising a HECT-RCC1 polypeptide in combination with at least one polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, a Gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4-like, and a clathrin.
- 2. An isolated protein complex comprising a HECT-RCC1 polypeptide and a Gag protein in combination with a polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4-like, and a clathrin.
- 3. An isolated protein complex comprising a VMSP polypeptide and a HIV Gag protein in combination with a polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4, and a clathrin.
- 4. An isolated protein complex comprising a HECT-WW polypeptide and a HIV Gag protein in combination with a polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4, and a clathrin.
- 5. The isolated protein complex of any one of claims 1 or 2, wherein said HECT-RCC1 polypeptide is Herc1.
- 6. The isolated protein complex of any one of claims 1 or 2, wherein said HECT-RCC1 polypeptide is Herc2.
- 7. The isolated protein complex of any one of claims 1 or 2, wherein said HECT-RCC1 polypeptide is Herc3.
- 8. The isolated protein complex of claim 3, wherein said VMSP is Nedd4.
- 9. The isolated protein complex of claim 4, wherein said HECT-WW is Nedd4.
- 10. The isolated protein complex of claim 3, wherein said VMSP is Herc1.
- 11. The isolated protein complex of claim 3, wherein said VMSP is Herc2.
- 12. The isolated protein complex of claim 3, wherein said VMSP is Herc3.
- 13. The isolated protein complex of any one of claims 1 or 2, wherein said Gag protein is an HIV gag protein.
- 14. The isolated protein complex of any one of claims 1 through 4, wherein said Gag protein comprises the Gag late domain.
- 15. The isolated protein complex of claim 14, wherein said Gag late domain is PTAP.
- 16. The isolated protein complex of claim 14, wherein said Gag late domain is PxxY.
- 17. The isolated protein complex of claim 14, wherein said Gag late domain is PxxL.
- 18. The isolated protein complex of claim 14, wherein said Gag late domain is PPxY.
- 19. The isolated protein complex of claim 14, wherein said Gag late domain is YxxL.
- 20. The isolated protein complex of claim 14, wherein said Gag late domain is PxxP.
- 21. A host cell comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a recombinant VMSP nucleic acid, and wherein the second nucleic acid comprises a recombinant nucleic acid encoding a Gag protein.
- 22. A host cell comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a recombinant HECT-WW nucleic acid, and wherein the second nucleic acid comprises a recombinant nucleic acid encoding a Gag protein.
- 23. A host cell comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a recombinant HECT-RCC1 nucleic acid, and wherein the second nucleic acid comprises a recombinant nucleic acid encoding a Gag protein.
- 24. The host cell of any one of claims 21 or 22, wherein said first nucleic acid is a Nedd4-like nucleic acid.
- 25. The host cell of any one of claims 21 or 23, wherein said first nucleic acid is a Herc1 nucleic acid.
- 26. The host cell of any one of claims 21 or 23, wherein said first nucleic acid is a Herc2 nucleic acid.
- 27. The host cell of any one of claims 21 or 23, wherein said first nucleic acid is a Herc3 nucleic acid.
- 28. The host cell of any one of claims 21, 22 or 23, wherein said Gag protein is an HIV gag protein.
- 29. The host cell of claim 28, wherein said Gag protein comprises the Gag late domain.
- 30. The host cell of claim 29, wherein said Gag late domain is PTAP.
- 31. The host cell of claim 29, wherein said Gag late domain is PxxY.
- 32. The host cell of claim 29, wherein said Gag late domain is PxxL.
- 33. The host cell of claim 29, wherein said Gag late domain is PPxY.
- 34. The host cell of claim 29, wherein said Gag late domain is YxxL.
- 35. The host cell of claim 29, wherein said Gag late domain is PxxP.
- 36. The host cell of claim 21, wherein the VMSP nucleic acid encodes a polypeptide comprising a polypeptide sequence at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 11-12 and 26-29 wherein the encoded polypeptide forms a complex with a Gag polypeptide.
- 37. The host cell of claim 22, wherein the HECT-WW nucleic acid encodes a polypeptide comprising a polypeptide sequence at least 95% identical to an amino acid sequence set forth in any one of SEQ ID Nos:11 and 12 and wherein the encoded polypeptide forms a complex with a Gag polypeptide.
- 38. The host cell of claim 23, wherein the HECT-RCC1 nucleic acid encodes a polypeptide comprising a polypeptide sequence at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NO: 26-29 and wherein the encoded polypeptide forms a complex with a Gag polypeptide.
- 39. A method for identifying modulators of protein complexes, comprising:
(i) forming a reaction mixture comprising
(a) a VMSP; and (b) a second polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, a gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4, Nedd4-like, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) determining the effect of said test agent for one or more activities selected from the group comprising (a) a change in the level of the protein complex, (b) a change in the enzymatic activity of the complex, or (c) where the reaction mixture is a whole cell, a change in the plasma membrane localization of the complex or a component thereof.
- 40. A method for identifying a test compound which inhibits or potentiates complex formation, comprising:
(i) forming a reaction mixture comprising
(a) a VMSP; and (b) a second polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, a gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4, Nedd4-like, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said VMSP to said second polypeptide; wherein a change in the binding of said VMSP to said second polypeptide in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said VMSP and said second polypeptide.
- 41. A method for identifying a test compound which inhibits or potentiates complex formation, comprising:
(i) forming a reaction mixture comprising
(a) a HECT-WW; and (b) a second polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, a Gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4, Nedd4-like, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said HECT-WW to said second polypeptide; wherein a change in the binding of said HECT-WW to said second polypeptide in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said HECT-WW and said second polypeptide.
- 42. A method for identifying a test compound which inhibits or potentiates complex formation, comprising:
(i) forming a reaction mixture comprising
(a) a HECT-RCC I; and (b) a second polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, a gag protein, a Gag late domain, PI3 K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC 1, HERC2, HERC3, Nedd4, Nedd4-like, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said HECT-RCC1 to said second polypeptide; wherein a change in the binding of said HECT-RCC1 to said second polypeptide in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said HECT-RCC1 and said second polypeptide.
- 43. A method for identifying a test compound which inhibits or potentiates complex formation, comprising:
(i) forming a reaction mixture comprising
(i) a Nedd4; and (ii) a second polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, an HIV gag protein, an HIV Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4-like, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said Nedd4 to said second polypeptide; wherein a change in the binding of said Nedd4 to said second polypeptide in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said Nedd4 and said second polypeptide.
- 44. A method for identifying a test compound which inhibits or potentiates complex formation, comprising:
(i) forming a reaction mixture comprising:
(a) a Nedd4; and (b) an HIV Gag protein; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said Nedd4 to said HIV Gag protein; wherein a change in the binding of said Nedd4 to said HIV Gag protein in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said Nedd4 and said HIV Gag protein.
- 45. A method for identifying a test compound which inhibits or potentiates complex formation, comprising:
(i) forming a reaction mixture comprising:
(a) a first polypeptide selected from the group consisting of Herc 1, Herc2, and Herc3; and (b) a second polypeptide selected from the group consisting of: HECT-WW, HECT-RCC1, a gag protein, a Gag late domain, PI3K, actin, myosin, Hsp60, Hsp70, Hsp90, STAM1, STAM2A, STAM2B, VHS-UIM, a GTPase, an E2 enzyme, tsg101, a cullin, HERC1, HERC2, HERC3, Nedd4, Nedd4-like, and a clathrin; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said first polypeptide to said second polypeptide; wherein a change in the binding of said first polypeptide to said second polypeptide in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said first polypeptide and said second polypeptide.
- 46. A method for identifying a test compound which inhibits or potentiates complex formation, comprising:
(i) forming a reaction mixture comprising:
(a) a first polypeptide selected from the group consisting of Herc1, Herc2, and Herc3; and (b) a Gag protein; (ii) contacting said reaction mixture with a test agent, and (iii) detecting binding of said first polypeptide to said Gag protein; wherein a change in the binding of said first polypeptide to said Gag protein in the presence of the test compound, relative to binding in the absence of the test compound, indicates that said test compound potentiates or inhibits complex formation between said first polypeptide and said Gag protein.
- 47. A method for inhibiting infection in a subject in need thereof, comprising administering an effective amount of an agent that inhibits the binding of a HECT-WW polypeptide to an gag protein.
- 48. A method for inhibiting infection in a subject in need thereof, comprising administering an effective amount of an agent that inhibits the binding of a HECT-RCC1 polypeptide to a gag protein.
- 49. The method of any one of claim 47 or claim 48, wherein said agent is selected from the group comprising a small molecule, a antibody, and a peptide.
- 50. The method of any one of claim 47 or claim 48, wherein the Gag protein is an HIV Gag protein.
- 51. The method of claim 50, wherein the Gag polypeptide is HIV p24.
- 52. An isolated antibody, or fragment thereof, specifically immunoreactive with an epitope of a VMSP polypeptide, which disrupts the interaction between said VMSP and a viral maturation scaffolding polypeptide-associating polypeptide (VMSP-AP), wherein said VMSP is encoded by a nucleic acid sequence which hybridizes under stringent conditions, to a nucleotide sequence set forth in any one of SEQ ID Nos: 7-8 and 19-22.
- 53. An isolated antibody, or fragment thereof, wherein said antibody is specifically immunoreactive with an epitope of a VMSP, which disrupts the interaction between said VMSP and a viral maturation scaffolding polypeptide-associating polypeptide (VMSP-AP), wherein said VMSP comprises an amino acid sequence at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID Nos: 15-16 and 26-30.
- 54. An isolated antibody of claim 52, which antibody is specifically immunoreactive with an epitope of a HECT-WW polypeptide, which disrupts the interaction between said HECT-WW polypeptide with a VMSP-AP, wherein said HECT-WW polypeptide is encoded by a nucleic acid sequence which hybridizes under stringent conditions, to a nucleotide sequence of any one of SEQ ID Nos: 7-8
- 55. An isolated antibody, or fragment thereof, wherein said antibody is specifically immunoreactive with an epitope of a HECT-WW polypeptide, which disrupts the interaction between said HECT-WW polypeptide with a VMSP-AP, wherein said HECT-WW polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID Nos: 15-16.
- 56. An isolated antibody, or fragment thereof, wherein said antibody is specifically immunoreactive with an epitope of a HECT-RCC1 polypeptide, which disrupts the interaction between said HECT-RCC1 polypeptide with a VMSP-AP, wherein said HECT-RCC1 polypeptide is encoded by a nucleic acid sequence which hybridizes under stringent conditions, to a nucleotide sequence in any one of SEQ ID Nos: 19-22.
- 57. An isolated antibody, or fragment thereof, wherein said antibody is specifically immunoreactive with an epitope of a HECT-RCC1 polypeptide, which disrupts the interaction between said HECT-RCC1 polypeptide with a VMSP-AP, wherein said HECT-RCC1 polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID Nos: 26-29.
- 58. The antibody of any one of claims 52-57, wherein said antibody is a monoclonal antibody.
- 59. The antibody of any one of claims 52-57, wherein said antibody is a Fab fragment.
- 60. The antibody of any one of claims 52-57, wherein said antibody is labeled with a detectable label.
- 61. The antibody of any one of claims 52-57, wherein said VMSP-AP is a gag polypeptide.
- 62. The antibody of any one of claims 52-57, wherein said VMSP-AP is an HIV gag polypeptide.
- 63. A purified preparation of polyclonal antibodies, or fragments thereof, wherein said antibodies are immunoreactive with an epitope of a VMSP, which epitope interacts with a VMSP-AP, wherein said VMSP comprises an amino acid sequence at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID Nos: 15-16 and 26-29.
- 64. A kit for detecting a VMSP polypeptide protein comprising (i) isolated anti-VMSP antibodies, or fragment thereof, specifically immunoreactive with an epitope of a VMSP, which epitope interacts with a VMSP-AP, and (ii) a detectable label for detecting said anti-VMSP antibody in immunoclomplexes with said VMSP polypeptide.
- 65. A host cell comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a recombinant HECT-WW nucleic acid, and wherein the second nucleic acid comprises a recombinant nucleic acid encoding a HIV Gag protein.
- 66. A method for inhibiting infection in a subject in need thereof, comprising administering an effective amount of an agent that inhibits the binding of a HECT-WW polypeptide to an HIV gag protein.
- 67. A method of inhibiting budding in a subject in need thereof, comprising administering an effective amount of an agent that inhibits the binding of a Herc1, Herc2, and Herc3, to a VMSP-AP.
- 68. The method of claim 67, wherein said VMSP-AP is a gag protein.
- 69. The method of claim 68, wherein said gag protein is an HIV gag protein.
- 70. The method of claim 69, wherein said Gag protein comprises the Gag late domain.
- 71. The method of claim 70, wherein said Gag late domain is PTAP.
- 72. The method of claim 70, wherein said Gag late domain is PxxY.
- 73. The method of claim 70, wherein said Gag late domain is PxxL.
- 74. The method of claim 70, wherein said Gag late domain is PPxY.
- 75. The method of claim 70, wherein said Gag late domain is YxxL.
- 76. The method of claim 70, wherein said Gag late domain is PxxP.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 60/275,224, filed Mar. 12, 2001, U.S. Provisional Application No. 60/308,958, filed Jul. 31, 2001, and U.S. Provisional Application No. 60/340,170, filed Dec. 7, 2001.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60275224 |
Mar 2001 |
US |
|
60308958 |
Jul 2001 |
US |
|
60340170 |
Dec 2001 |
US |