Cell-based method and assay for measuring the infectivity and drug sensitivity of immunodeficiency virus

Abstract

Methods and reagents for the capture of primary HIV are provided. A cell line expressing CCR5, CXCR4 and CD4 receptors binds and is infected by primary HIV. The cell line contains a marker gene sequence, the marker gene sequence expressed in near linear quantities over at least two orders of magnitude in response to HIV infection. Primary HIV is amplified to create a primary virus stock through insertion of an amplicon gene into the receptor expressing cell line. HIV amplification occurs rapidly and is operative with noninfectious HIV through amplification in the presence of an infectivity complement. The present invention is useful in determining host HIV titer, drug sensitivity, HIV amplification, gene sequencing and co-receptor utilization.

1 . A method for determining the sensitivity of an HIV virus to an antiviral compound comprising: 
 a) providing an immortalized cell characterized as expressing CCR 5 , CXCR4, and CD4 receptors and having stably incorporated a marker gene operably linked to a promoter, wherein expression of the marker gene occurs upon HIV infection of the immortalized cell;    b) providing the immortalized cell a first composition comprising at least one anti-viral compound;    c) contacting said immortalized cell with a second composition comprising at least one HIV virus; and,    d) assaying for marker gene expression.    
     
     
         2 . The method of  claim 1 , wherein said marker gene encodes luciferase, β-galactosidase, green fluorescent protein, or a polypeptide that infers antibiotic resistance.  
     
     
         3 . The method of  claim 1 , wherein said promoter comprises the HIV-1 LTR or HIV-2 LTR.  
     
     
         4 . The method of  claim 1 , wherein said HIV-1 virus is a primary HIV-1 virus.  
     
     
         5 . The method of  claim 1 , wherein said anti-viral compound is selected from the group consisting of AZT, 3TC, Nevaripine, and an antibody.  
     
     
         6 . The method of  claim 1 , wherein the anti-viral compound affects early stages of the viral life cycle.  
     
     
         7 . The method of  claim 6 , wherein said anti-viral compound affects the activity of Reverse Transcriptase, Integrase, or Env.  
     
     
         8 . The method of  claim 1 , wherein said anti-viral compound affects late stages of the viral life cycle.  
     
     
         9 . The method of  claim 8 , wherein said anti-viral compound affects the activity of Gag or protease.  
     
     
         10 . The method of  claim 1 , wherein said anti-viral compound is indinavir.  
     
     
         11 . The method of  claim 1 , wherein expression of the marker gene quantitates the sensitivity of the HIV virus to the antiviral compound.  
     
     
         12 . The method of  claim 6 , wherein expression of the marker gene quantitates the sensitivity of the HIV virus to the antiviral compound.  
     
     
         13 . The method of  claim 8 , wherein expression of the marker gene quantitates the sensitivity of the HIV virus to the antiviral compound.  
     
     
         14 . An immortalized cell characterized as expressing CCR5, CXCR4, and CD4 receptors and having stably incorporated an amplicon gene operably linked to a promoter.  
     
     
         15 . A method for amplifying HIV virus comprising: 
 a) providing an immortalized cell characterized as expressing CCR5, CXCR4, and CD4 receptors and having stably incorporated an amplicon gene operably linked to a promoter active in said immortalized cell; and,    b) contacting said immortalized cell with a composition comprising at least one HIV viral isolate.    
     
     
         16 . The method of  claim 15 , wherein said HIV viral isolate is a primary HIV-1 viral isolate.  
     
     
         17 . The method of  claim 15 , wherein said promoter is a constitutive or an inducible promoter.  
     
     
         18 . The method of  claim 15 , wherein said promoter is a CMV promoter.  
     
     
         19 . The method of  claim 15 , wherein the immortalized cell further has stably incorporated a marker gene operably linked to a second promoter, wherein expression of the marker gene occurs upon HIV infection of the cell.  
     
     
         20 . The method of  claim 19 , wherein said marker gene encodes luciferase, β-galactosidase, green fluorescent protein, or polypeptide that infers antibiotic resistance.  
     
     
         21 . The method of  claim 16 , further comprising isolating said HIV virus.  
     
     
         22 . The method of  claim 21 , further comprising: 
 a) contacting a second immortalized cell with the isolated HIV virus, wherein said second immortalized cell is characterized by expressing CCR5, CXCR4, and CD4 receptors and having stably incorporated a marker gene operably linked to a second promoter, wherein expression of the marker gene occurs upon HIV infection of the immortalized cell; and,    b) assaying for marker gene expression.