Claims
- 1. An isolated nucleic acid comprising a nucleic acid sequence encoding a polypeptide that comprises four SH3 domains and is at least 90% identical to SEQ ID NO:2, or a complement thereof.
- 2. The nucleic acid of claim 1, wherein the polypeptide is at least 95% identical to SEQ ID NO:2.
- 3. The isolated nucleic acid of claim 1, wherein the polypeptide sequence additionally comprises a RING domain.
- 4. The isolated nucleic acid of claim 1, wherein the nucleic acid mitigates a POSH loss of function phenotype in a cell.
- 5. The isolated nucleic acid of claim 4, wherein the POSH loss of function phenotype is a decrease in HIV virus like particle production.
- 6. An isolated nucleic acid comprising a nucleic acid sequence encoding a polypeptide comprising at least four SH3 domains, which isolated nucleic acid sequence hybridizes under stringent conditions to a nucleic acid selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, a complement of SEQ ID NO:1 and a complement of SEQ ID NO:3.
- 7. An isolated nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO:1, a complement of SEQ ID NO:1, SEQ ID NO:3 and a complement of SEQ ID NO:3.
- 8. The nucleic acid sequence of any of claims 1-7, wherein the sequence encoding the polypeptide is disrupted by at least one intron.
- 9. The nucleic acid of any of claims 1-7, further comprising a transcriptional regulatory sequence operably linked to the nucleotide sequence so as to render the nucleic acid suitable for use as an expression vector.
- 10. An expression vector comprising a nucleic acid of any of claims 1-7.
- 11. The expression vector of claim 10, which replicates in at least one of a prokaryotic cell and a eukaryotic cell.
- 12. A host cell transfected with the expression vector of claim 10.
- 13. The host cell of claim 12, wherein the cell expresses a POSH polypeptide.
- 14. A purified POSH polypeptide expressed by the host cell of claim 13.
- 15. A method of producing a recombinant POSH polypeptide comprising culturing the cell of claim 13 in a cell culture medium suitable for expression of the POSH polypeptide.
- 16. The method of claim 15, further comprising a purification procedure to increase the purity of the POSH polypeptide.
- 17. A nucleic acid encoding a fusion protein, wherein the nucleic acid comprises a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence is a nucleic acid of any of claims 1-7, and wherein the second nucleic acid sequence encodes a heterologous polypeptide, and wherein the first nucleic acid sequence and the second nucleic acid sequence are positioned to encode a fusion protein.
- 18. The nucleic acid of claim 17, wherein the heterologous polypeptide is (i) a detectable label or (ii) a matrix-binding domain.
- 19. A ribonucleic acid comprising between 5 and 1000 consecutive nucleotides of a nucleic acid sequence that is at least 90% identical to a sequence of SEQ ID NO:1 and/or 3 or a complement thereof and which, when introduced into a cell, decreases the level of a POSH mRNA and/or a POSH polypeptide.
- 20. The ribonucleic acid of claim 19, comprising between 5 and 1000 consecutive nucleotides of SEQ ID NO:1 and/or 3, or a complement thereof.
- 21. The ribonucleic acid of claim 19, comprising between 15 and 30 consecutive nucleotides of SEQ ID NO: 1 and/or 3 or a complement thereof.
- 22. The ribonucleic acid of claim 19, wherein the ribonucleic acid is a double stranded ribonucleic acid.
- 23. The ribonucleic acid of claim 19, which is selected from the consisting of an siRNA and a ribozyme.
- 24. The ribonucleic acid of claim 19, comprising a sequence selected from the group consisting of any of SEQ ID Nos:15, 16, 18, 19, 21, 22, 24 and 25.
- 25. A composition comprising the ribonucleic acid of claim 19 and an additional component selected from the group consisting of: a liposome and a pharmaceutically effective excipient.
- 26. The composition of claim 25 formulated for topical administration.
- 27. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
a) an amino acid sequence at least 91% identical to SEQ ID NO:2 b) an amino acid sequence encoded by a nucleic acid that hybridizes under high stringency conditions to a nucleic acid of any one of SEQ ID Nos: 1 or 3, wherein optionally (i) said polypeptide interacts with a POSH-AP and/or (ii) down regulation of said polypeptide decreases viral maturation.
- 28. The isolated polypeptide of claim 27, wherein said down regulation of said polypeptide decreases viral maturation of a virus selected from the group consisting of an envelope virus, a retroid virus and an RNA virus.
- 29. The isolated polypeptide of claim 28, wherein
a) the retroid virus is a lentivirus; b) the retroid virus is an HIV1 lentivirus; c) the RNA virus is a filovirus; or d) the RNA virus is an ebola filovirus.
- 30. The isolated polypeptide of claim 27, wherein said down regulation of said polypeptide decreases viral maturation of a PTAP or PPEY motif containing virus.
- 31. The isolated polypeptide of any one of claims 27-30, wherein the polypeptide comprises four SH3 domains and/or a RING domain.
- 32. The polypeptide of any one of claims 27-30, wherein said polypeptide is purified to at least 80% by dry weight.
- 33. The polypeptide of any one of claims 27-30, wherein said polypeptide interacts with a POSH-AP selected from the group consisting of: an E2, a POSH polypeptide, a ubiquitin, and a GTPase.
- 34. The polypeptide of claim 33, wherein the GTPase is Rac.
- 35. The polypeptide of claim 33, wherein the Rac is Rac1.
- 36. The polypeptide of any of claims 27-30, wherein the polypeptide is a fusion protein.
- 37. The polypeptide of claim 36, wherein said fusion protein is functional in a two-hybrid screening assay.
- 38. An isolated antibody, or fragment thereof, specifically immunoreactive with an epitope of a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID No:26, and SEQ ID NO:30.
- 39. The antibody of claim 38, which disrupts the interaction between a polypeptide of SEQ ID NO:2 and a POSH-AP.
- 40. The antibody of claim 38, wherein said antibody is selected from the group consisting of: a polyclonal antibody, a monoclonal antibody, an Fab fragment and a single chain antibody.
- 41. The antibody of claim 38, wherein said antibody is labeled with a detectable label.
- 42. The antibody of claim 39, wherein the POSH-AP is selected from the group consisting of: an E2, a POSH polypeptide, a ubiquitin, and a GTPase.
- 43. The polypeptide of claim 43, wherein the GTPase is Rac and wherein the Rac is optionally Rac1.
- 44. A kit for detecting a human POSH polypeptide comprising (i) an antibody of any one of claims 38-39, and (ii) a detectable label for detecting said antibody.
- 45. A method for identifying an antiviral agent comprising:
providing a POSH polypeptide and a test agent; and identifying a test agent that interacts with the POSH polypeptide.
- 46. The method of claim 45, wherein the POSH polypeptide is selected from the group consisting of SEQ ID NOS:2, 5, 7, 9 and 11 and fragments comprising at least 20 consecutive amino acids of anv of SEQ ID Nos:2, 5, 7, 9, and 11.
- 47. The method of claim 45, wherein the POSH polypeptide is expressed in a cell.
- 48. The method of claim 45, wherein the POSH polypeptide is a purified polypeptide.
- 49. The method of any of claims 46-48, wherein the POSH polypeptide comprises a domain selected from the group consisting of: an SH3 domain and a RING domain.
- 50. The method of claim 49, wherein the test agent binds to a domain selected from the group consisting of: an SH3 domain and a RING domain.
- 51. The method of claim 48, wherein the test agent is a polypeptide, an antibody, a small molecule or a peptidomimetic.
- 52. The method of claim 50, wherein a test agent that interacts with POSH decreases the maturation of a virus containing the PTAP or PPEY motif.
- 53. A method for identifying an antiviral agent comprising:
providing a POSH-mediated viral release polypeptide and a test agent; and identifying a test agent that interacts with the POSH-mediated viral release polypeptide.
- 54. The method of claim 53, wherein the POSH-mediated viral release polypeptide is selected from the group consisting of POSH, POSH-multimers, a polypeptide comprising a RING domain and a SH3 domain, Brca1, Bard1, Nef, Rac, Rac1, PAK1, PAK2, PAK family, Vav,Cdc42, P13K, Nedd4, src, src family polypeptides, gag, tsg101, VASP, RNB6, WASP, N-WASP, KIAA0674.
- 55. The method of claim 54, wherein the test agent is a polypeptide, an antibody, a small molecule or a peptidomimetic.
- 56. A method for identifying an antiviral agent comprising:
providing a POSH nucleic acid and a test agent; and identifying a test agent that binds to the POSH nucleic acid.
- 57. The method of claim 59, wherein the POSH nucleic acid is selected from the group consisting of SEQ ID Nos:1, 3, 4, 6, 8, and 10.
- 58. The method of claim 59, wherein the test agent is a ribonucleic acid, an antisense oilgonucleotide, a RNAi construct, a DNA enzyme, and a ribozyme.
- 59. The method of claim 59, wherein binding of the test agent to said POSH nucleic acid decreases the level of a POSH transcript.
- 60. A method for identifying an antiviral agent comprising:
providing a POSH-mediated viral release nucleic acid and a test agent; and identifying a test agent that interacts with the POSH-mediated viral release nucleic acid.
- 61. The method of claim 60, wherein the POSH-mediated viral release nucleic acid is selected from the group consisting of POSH, POSH-multimers, a nucleic acid encoding polypeptide comprising a RING domain and a SH3 domain, Brca1, Bard1, Nef, Rac, Racl, PAK1, PAK2, PAK family, Vav,Cdc42, P13K, Nedd4, src, src family polypeptides, gag, tsg101, VASP, RNB6, WASP, N-WASP, KIAA0674.
- 62. The method of claim 61, wherein the test agent is a ribonucleic acid, an antisense oilgonucleotide, a RNAi construct, a DNA enzyme, and a ribozyme.
- 63. The method of any of claims 45-62, further comprising:
administering a composition comprising the test agent to a cell transfected with At least a portion of a viral genome; and measuring the effect of the test agent on the production of viral or virus-like particles.
- 64. The method of any of claims 48-62, wherein the antiviral agent is effective against a virus selected from the group consisting of: an envelope virus, a retroid virus and a RNA virus.
- 65. The method of claim 64, wherein:
a) the retroid virus is a lentivirus; b) the retroid virus is an HIV1 lentivirus; c) the RNA virus is a filovirus; or d) the RNA virus is an ebola filovirus.
- 66. A method for identifying an antiapoptotic agent comprising:
providing a POSH polypeptide and a test agent, wherein the POSH polypeptide is at least 80% identical to SEQ ID NO:2; and identifying a test agent that binds to the POSH polypeptide.
- 67. The method of claim 66, wherein the test agent binds to a domain selected from the group consisting of: an SH3 domain and a RING domain.
- 68. The method of claim 66, wherein the POSH polypeptide is expressed in a cell.
- 69. The method of claim 66, wherein the POSH polypeptide is a purified polypeptide.
- 70. A method for identifying an antiapoptotic agent comprising identifying a test agent that binds to a POSH nucleic acid.
- 71. The method of claim 70, wherein the test agent is a ribonucleic acid.
- 72. The method of any of claims 66-71, further comprising:
administering a composition comprising the test agent to a cell overexpressing a POSH polypeptide; and measuring the effect of the test agent on POSH-induced apoptosis.
- 73. A method for inhibiting infection in a subject in need thereof, comprising administering an effective amount of an agent that inhibits a POSH activity.
- 74. The method of claim 73, wherein the agent inhibits the Ubgiquitin ligase activity of the POSH polypeptide.
- 75. The method of claim 73, wherein the agent is selected from the group consisting of a small molecule, a antibody, a fragment of an antibody, a peptidomimetic, and a polypeptide.
- 76. The method of claim 73, wherein the agent inhibits the interaction between a POSH polypeptide and a POSH-AP.
- 77. The method of claim 75, wherein the POSH-AP is selected from the group consisting of: an E2, a POSH polypeptide, a ubiquitin, and a GTPase.
- 78. The method of claim 77, wherein the GTPase is Rac.
- 79. The method of claim 78, wherein the Rae and wherein the Rac1.
- 80. The method of claim 73, wherein the agent selected from the group consisting of an antisense oligonucleotide, an RNAi construct, a DNA enzyme, and a ribozyme.
- 81. The method of claim 80, wherein the RNAi construct is selected from the group consisting of any one of SEQ ID Nos; 15, 18, 18, 19, 21, 22, 24, and 25.
- 82. The method of claim 80, wherein the agent decreases the level of POSH mRNA.
- 83. A non-human transgenic animal comprises a plurality of cells containing one or more recombinant constructs of a POSH gene, wherein expression of said POSH gene mitigates a POSH loss of function phenotype.
- 84. The non-human transgenic animal of claim 83, wherein said one or more recombinant constructs are introduced into said animal or an ancestor of said animal, at an embryonic stage.
- 85. An isolated cell of the transgenic animal of claim 83.
- 86. The cell of claim 85, wherein the cell is a germ cell.
- 87. The cell of claim 85, wherein the cell is a somatic cell.
- 88. The non-human transgenic animal of claim 83, wherein all germ cells and somatic cells contain one or more recombinant constructs of said POSH gene.
- 89. The non-human transgenic animal of claim 84, wherein said recombinant construct is introduced into the embryo by microinjection, electroporation, or lipofection.
- 90. The non-human transgenic animal of claim 83, wherein the non-human animal is selected from the group consisting of a mouse, rat, pig, sheep, goat, rabbit, cow, and a monkey.
- 91. The non-human animal of claim 83, wherein the POSH gene is human POSH transgene.
- 92. A method of constructing the non-human transgenic animal of claim 83, comprising introducing a POSH transgene, into a fertilized egg and implanting said fertilized egg.
- 93. The non-human transgenic animal of claim 83, wherein the POSH loss of function phenotype is a decrease in the production of virus/viral like particles.
- 94. A method of screening for a substance to be used for treating a viral infection, comprising administering a test compound to the non-human animal of claim 83, and assaying efficacy of said test compound in potentiating the POSH loss of function phenotype.
- 95. A method of evaluating the anti-viral potential of an agent comprising (i) infecting the transgenic non-human animal of claim 83 with a virus selected from the group consisting of a envelope virus an retroid virus and a RNA virus; (ii) contacting transgenic non-human animal of step (i) with a test agent, and (iii) comparing the production of viral like particle in a sample from the treated animal with the production of viral like particles in a sample from an untreated transgenic animal or a transgenic animal treated with a control agent, wherein the difference in the production of virus like particles in the treated animal, relative to the production of virus like particles in the absence of treatment or treatment with a control agent, indicates the anti-viral potential of the test compound.
- 96. A method of evaluating an anti-viral activity of a test compound, comprising:
(i) infecting a non-human animal transgenic animal of claim 83, having germline and somatic cells expressing a human POSH transgene, or a sample of cells derived therefrom with a virus selected from the group consisting of a envelope virus, a retroid virus or an RNA virus; (ii) contacting the non-human transgenic animal or the sample of cells of step (i) with a test agent; and (iii) determining the production of virus like particles in a specimen from the transgenic animal or in the sample of cells, wherein a statistically significant decrease in the production of virus like particles, relative to the production of virus like particles in the absence of the test agent, indicates the test compound is a potential anti-viral agent.
- 97. The method of any of claims 94-96, wherein:
a) the retroid virus is a lentivirus; b) the retroid virus is an HIV1 lentivirus; c) the RNA virus is a filovirus; or d) the RNA virus is an ebola filovirus.
- 98. A method of inhibiting viral maturation comprising inhibiting a ubiquitin-related activity of a POSH polypeptide.
- 99. The method of claim 98, wherein the POSH polypeptide is selected from the group consisting of SEQ ID NOS:2, 5, 7, 9 and 11 and fragments comprising at least 20 consecutive amino acids of any of SEQ ID Nos:2, 5, 7, 9, and 11.
- 100. The method of claim 98, comprising inhibiting an activity of the RING domain of the POSH polypeptide.
- 101. The method of claim 98, wherein viral maturation is inhibited by administering an agent selected from the group consisting of a small molecule, a antibody, a peptidomimetic, and a polypeptide.
- 102. The method of claim 101, wherein the agent is an antibody or a fragment thereof, specifically immunoreactive with an epitope of SEQ ID No: 30.
- 103. The method of claim 98, wherein viral maturation is inhibited by administering an agent selected from the group consisting of a an antisense oligonucleotide, an RNAi construct, a DNA enzyme, and a ribozyme.
- 104. The method of claim 103, wherein the RNAi construct is selected from the group consisting of anyone of SEQ ID Nos:15, 18, 18, 19, 21, 22, 24, and 25.
- 105. The method of claim 98, comprising inhibiting viral maturation of a virus selected from the group consisting of a envelope virus, retroid virus or a RNA virus.
- 106. The method of claim 105, wherein:
a) the retroid virus is a lentivirus; b) the retroid virus is an HIV1 lentivirus; c) the RNA virus is a filovirus; or d) the RNA virus is an ebola filovirus.
- 107. A method for testing a ubiquitin-related activity of a POSH polypeptide comprising:
forming a mixture compatible with the ubiquitin-related activity comprising: a ubiquitin; an E1; an E2; and a POSH polypeptide; and detecting whether said ubiquitin binds to said POSH polypeptide.
- 108. The method of claim 107, wherein the POSH polypeptide is selected from the group consisting of SEQ ID NOS:2, 5, 7, 9, 11 and 26.
- 109. The method of claim 107, wherein the ubiquitin is detectably labeled.
- 110. The method of claim 107, wherein the POSH polypeptide is detectably labeled.
- 111. The method of claim 109 or 110, wherein said label is selected from the group consisting of a radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
- 112. An assay for identifying an inhibitor of a ubiquitin-related activity of a POSH polypeptide, comprising:
(i) providing a mixture comprising a ubiquitin; E2; and a POSH polypeptide, under conditions which promote ubiquitination of the POSH polypeptide; (ii) contacting the ubiquitin-conjugating system with a test agent; (iii) measuring a level of ubiquitination of the POSH polypeptide in the presence of the test agent; and (iv) comparing the measured level of ubiquitination in the presence of the test agent with a suitable reference, wherein a decrease in ubiquitination of the POSH polypeptide in the presence of the candidate agent is indicative of an inhibitor of ubiquitination of the regulatory protein.
- 113. The assay of claim 112, wherein the ubiquitin is provided in a form selected from the group consisting of:
an unconjugated ubiquitin, in which case the ubiquitin-conjugating system further comprises an E1 and adenosine triphosphate; an activated E1:ubiquitin conjugate; and an activated E2:ubiquitin thioester complex.
- 114. The assay of claim 112, wherein the ubiquitin is detectably labeled.
- 115. The assay of claim 112, wherein the POSH polypeptide is detectably labeled.
- 116. A therapeutic composition comprising a inhibitor of any one of a POSH polypeptide, a polypeptide set forth in SEQ ID No: 26, and a polypeptide set forth in SEQ ID No: 30, and a pharmaceutically acceptable excipient.
- 117. The therapeutic composition of claim 116, wherein the inhibitor is selected from the group consisting of a small molecule, a antibody, a polypeptide, and a peptidomimetic.
- 118. The therapeutic composition of claim 116, wherein the inhibitor disrupts the interaction between a POSH polypeptide and POSH-AP and/or inhibits a ubiquitin-related activity of a POSH polypeptide.
- 119. The therapeutic composition of claim 116, wherein the inhibitor is a polypeptide of SEQ ID NO: 30.
- 120. The therapeutic composition of claim 116, wherein the inhibitor is selected from the group consisting of a antisense oligonucleotide, a DNA enzyme, a RNAi construct, and a ribozyme.
- 121. The therapeutic composition of claim 116, wherein the RNAi construct is selected from the group consisting of any one of SEQ ID Nos;15, 18, 18, 19, 21, 22, 24, and 25.
- 122. A composition comprising a POSH polypeptide and Ubiquitin.
- 123. A POSH polypeptide-Ubiquiutin conjugate.
- 124. A complex comprising a POSH polypeptide and a POSP-AP.
- 125. A method of inhibiting viral infection comprising administering an agent to a subject in need thereof wherein said agent inhibits POSH-mediated viral release.
- 126. The method of claim 125, wherein the agent inhibits a POSH-mediated viral release polypeptide.
- 127. The method of claim 126, wherein the POSH-mediated viral release polypeptide is selected from the group consisting of POSH, POSH-multimers, a nucleic acid encoding polypeptide comprising a RING domain and a SH3 domain, Brca1, Bard1, Nef, Rac, Rac1, PAK1, PAK2, PAK family, Vav,Cdc42, P13K, Nedd4, src, src family polypeptides, gag, tsg101, VASP, RNB6, WASP, N-WASP, KIAA0674.
- 128. A method of identifying targets for therapeutic intervention comprising identifying a polypeptide that associates with anyone of a POSH polypeptide or a POSH-mediated viral release polypeptide.
- 129. The method of claim 128, wherein the POSH-mediated viral release polypeptide is selected from the group consisting of POSH, POSH-multimers, a nucleic acid encoding polypeptide comprising a RING domain and a SH3 domain, Brca1, Bard1, Nef, Rac, Rac1, PAK1, PAK,2, PAK family, Vav,Cdc42, P13K, Nedd4, src, src family polypeptides, gag, tsg101, VASP, RNB6, WASP, N-WASP, KIAA0674.
- 130. A method for evaluating the anti-viral potential of a compound comprising:
forming a mixture comprising a ubiquitin; an E1; an E2; and a POSH polypeptide; adding a test agent; and detecting ubiquitin-ligase activity of said POSH polypeptide wherein a compound that decreases the ligase activity of said POSH polypeptide is a potential anti-viral agent.
RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of U.S. Provisional Applications No. 60/345,846, filed Nov. 9, 2001, and No. 60/364,530, filed Mar. 15, 2002, the specifications of which are hereby incorporated by reference in their entirety.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60345846 |
Nov 2001 |
US |
|
60364530 |
Mar 2002 |
US |