US2003027198A1PendingUtilityA1

Mammalian genes involved in viral infection and tumor suppression

41
Priority: Apr 15, 1996Filed: Aug 27, 2002Published: Feb 6, 2003
Est. expiryApr 15, 2016(expired)· nominal 20-yr term from priority
Inventors:Donald RubinEdward OrganRaymond N. Dubois
G01N 2500/04A61K 38/00C12N 15/1034G01N 2333/005A61P 31/12C12Q 1/6883C12N 15/1082C07K 14/47C12Q 2600/158A61P 31/18C12N 15/1079C12N 15/1051G01N 33/575
41
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Claims

Abstract

The present invention provides methods of identifying cellular genes necessary for viral growth and cellular genes that function as tumor suppressors. Thus, the present invention provides nucleic acids related to and methods of reducing or preventing viral infection or cancer. The invention also provides methods of producing substantially virus-free cell cultures and methods for screening for additional such genes.

Claims

exact text as granted — not AI-modified
What is claimed is:  
     
         1 . An isolated nucleic acid comprising a nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83.  
     
     
         2 . An allelic variant or homolog of the nucleic acid of  claim 1 .  
     
     
         3 . An isolated nucleic acid encoding the protein encoded by the gene comprising the nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83.  
     
     
         4 . A host cell containing the nucleic acid of  claim 1 ,  2  or  3 .  
     
     
         5 . A nucleic acid that selectively hybridizes under stringent conditions with the nucleic acid of  claim 1 ,  2  or  3 .  
     
     
         6 . A nucleic acid having a region within an exon wherein the region has at least 50% homology with the nucleic acid of  claim 1 ,  2  or  3 .  
     
     
         7 . A nucleic acid having a region within an exon wherein the region has at least 60% homology with the nucleic acid of  claim 1 ,  2  or  3 .  
     
     
         8 . A nucleic acid having a region within an exon wherein the region has at least 70% homology with the nucleic acid of  claim 1 ,  2  or  3 .  
     
     
         9 . A nucleic acid having a region within an exon wherein the region has at least 80% homology with the nucleic acid of  claim 1 ,  2  or  3 .  
     
     
         10 . A nucleic acid having a region within an exon wherein the region has at least 90% homology with the nucleic acid of  claim 1 ,  2  or  3 .  
     
     
         11 . A nucleic acid having a region within an exon wherein the region has at least 95% homology with the nucleic acid of  claim 1 ,  2   3 .  
     
     
         12 . A protein encoded by the nucleic acid of claims  1 ,  2 ,  3 ,  5 ,  6 ,  7 ,  8 ,  9 ,  10  or  11 .  
     
     
         13 . A nucleic acid comprising a regulatory region of a gene comprising the nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83.  
     
     
         14 . A construct comprising a regulatory region of  claim 13 , wherein the regulatory region is functionally linked to a reporter gene.  
     
     
         15 . A method of identifying a cellular gene necessary for viral growth in a cell and nonessential for cellular survival, comprising 
 (a) transferring into a cell culture growing in serum-containing medium a vector encoding a selective marker gene lacking a functional promoter,    (b) selecting cells expressing the marker gene,    (c) removing serum from the culture medium,    (d) infecting the cell culture with the virus, and    (e) isolating from the surviving cells a cellular gene within which the marker gene is inserted, thereby identifying a gene necessary for viral growth in a cell and nonessential for cellular survival.    
     
     
         16 . A method of reducing or inhibiting a viral infection in a subject, comprising administering to the subject an amount of a composition that inhibits expression or functioning of a gene product encoded by a gene comprising the nucleic acid set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:1O, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or a homolog thereof, thereby treating the viral infection.  
     
     
         17 . The method of  claim 16 , wherein the composition comprises an antibody that binds a protein encoded by the gene.  
     
     
         18 . The method of  claim 16 , wherein the composition comprises an antibody that binds a receptor for a protein encoded by the gene.  
     
     
         19 . The method of  claim 16 , wherein the composition comprises an antisense RNA that binds an RNA encoded by the gene.  
     
     
         20 . The method of  claim 16 , wherein the composition comprises a nucleic acid functionally encoding an antisense RNA that binds an RNA encoded by the gene.  
     
     
         21 . A method of reducing or inhibiting a viral infection in a subject comprising mutating ex vivo in a selected cell from the subject an endogenous gene comprising the nucleic acid set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or a homolog thereof, to a mutated gene incapable of producing a functional gene product of the gene or to a mutated gene producing a reduced amount of a functional gene product of the gene, and replacing the cell in the subject, thereby reducing viral infection of cells in the subject.  
     
     
         22 . The method of  claim 21 , wherein the cell is a hematopoietic cell.  
     
     
         23 . A method of reducing or inhibiting a viral infection in a subject comprising mutating ex vivo in a selected cell from the subject an endogenous gene comprising a nucleic acid isolated by the method of  claim 15 , to a mutated gene incapable of producing a functional gene product of the gene or to a mutated gene producing a reduced amount of a functional gene product of the gene, and replacing the cell in the subject, thereby reducing viral infection of cells in the subject.  
     
     
         24 . The method of  claim 23 , wherein the virus is HIV.  
     
     
         25 . The method of  claim 23 , wherein the cell is a hematopoietic cell.  
     
     
         26 . A method of increasing viral infection resistance in a subject comprising mutating ex vivo in a selected cell from the subject an endogenous gene comprising a nucleic acid isolated by the method of  claim 15 , to a mutated gene incapable of producing a functional gene product of the gene or to a mutated gene producing a reduced amount of a functional gene product of the gene, and replacing the cell in the subject, thereby reducing viral infection of cells in the subject.  
     
     
         27 . The method of  claim 26 , wherein the virus is HIV.  
     
     
         28 . The method of  claim 26 , wherein the cell is a hematopoietic cell.  
     
     
         29 . A method of screening a compound for effectiveness in treating a viral infection, comprising administering the compound to a cell containing a cellular gene functionally encoding a gene product necessary for reproduction of the virus in the cell but not necessary for survival of the cell and detecting the level of the gene product produced, a decrease or elimination of the gene product indicating a compound effective for treating the viral infection.  
     
     
         30 . The method of  claim 29 , wherein the cellular gene comprises the nucleic acid set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, or a homolog thereof.  
     
     
         31 . The method of  claim 29 , wherein the cellular gene is a gene identified by the method of  claim 15 .  
     
     
         32 . A method of screening a compound for reducing or inhibiting a viral infection, comprising administering the compound to a cell containing the construct of  claim 14  and detecting the level of the reporter gene product produced, a decrease or elimination of the reporter gene product indicating a compound for reducing or inhibiting the viral infection.  
     
     
         33 . A purified mammalian serum protein having a molecular weight of between about 50 kD and 100 kD which resists inactivation in low pH and resists inactivation by chloroform extraction, which inactivates when boiled and inactivates in low ionic strength solution, and which when removed from a cell culture comprising cells persistently infected with reovirus selectively prevents survival of cells persistently infected with reovirus.  
     
     
         34 . A method of selectively eliminating, from an animal cell culture capable of surviving for a first period of time in the absence of serum, cells persistently infected with a virus, comprising propagating the cell culture in the absence of serum for a second time period which a persistently infected cell cannot survive without serum, thereby selectively eliminating from the cell culture cells persistently infected with the virus.  
     
     
         35 . The method of  claim 34 , wherein the second time period is from about three days to about ten days.  
     
     
         36 . The method of  claim 34 , further comprising transferring the cell culture from a first container to a second container.  
     
     
         37 . A method of selectively eliminating from a cell culture cells persistently infected with a virus, comprising propagating the cell culture in the absence of a functional form of the protein of  claim 33 .  
     
     
         38 . A method of reducing or inhibiting a viral infection in a subject, comprising administering to the subject an amount of a composition that inhibits functioning of a serum protein having a molecular weight of between about 50 kD and 100 kD which resists inactivation in low pH and resists inactivation by chloroform extraction, which inactivates when boiled and inactivates in low ionic strength solution, and which, when removed from a cell culture comprising cells persistently infected with the virus, prevents survival of cells persistently infected with the virus, thereby reducing or inhibiting the viral infection.  
     
     
         39 . The method of  claim 38 , wherein the composition comprises an antibody that binds the serum protein.  
     
     
         40 . The method of  claim 38 , wherein the composition comprises an antisense RNA that binds an RNA encoded by the gene.  
     
     
         41 . A method of identifying a cellular gene that can suppress a malignant phenotype in a cell, comprising 
 (a) transferring into a cell culture incapable of growing well in soft agar a vector encoding a selective marker gene lacking a functional promoter,    (b) selecting cells expressing the marker gene, and    (c) isolating from selected cells which are capable of growing in agar a cellular gene within which the marker gene is inserted, thereby identifying a gene that can suppress a malignant phenotype in a cell.    
     
     
         42 . A method of identifying a cellular gene that can suppress a malignant phenotype in a cell, comprising 
 (a) transferring into a cell culture of non-transformed cells a vector encoding a selective marker gene lacking a functional promoter,    (b) selecting cells expressing the marker gene, and    (c) isolating from selected and transformed cells a cellular gene within which the marker gene is inserted, thereby identifying a gene that can suppress a malignant phenotype in a cell.    
     
     
         43 . A method of screening for a compound for suppressing a malignant phenotype in a cell comprising administering the compound to a cell containing a cellular gene functionally encoding a gene product involved in establishment of a malignant phenotype in the cell and detecting the level of the gene product produced, a decrease or elimination of the gene product indicating a compound effective for suppressing the malignant phenotype.  
     
     
         44 . A method of suppressing a malignant phenotype in a cell in a subject, comprising administering to the subject an amount of a composition that inhibits expression or functioning of a gene product encoded by a gene comprising the nucleic acid set forth in SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82 or SEQ ID NO:83, or a homolog thereof, thereby suppressing a malignant phenotype.  
     
     
         45 . The method of  claim 44 , wherein the composition comprises an antibody that binds a protein encoded by the gene.  
     
     
         46 . The method of  claim 44 , wherein the composition comprises an antibody that binds a receptor for a protein encoded by the gene.  
     
     
         47 . The method of  claim 44 , wherein the composition comprises an antisense RNA that binds an RNA encoded by the gene.  
     
     
         48 . The method of  claim 44 , wherein the composition comprises a nucleic acid functionally encoding an antisense RNA that binds an RNA encoded by the gene.

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