Compositions and Methods for Evaluating Viral Receptor/Co-Receptor Usage and Inhibitors of Virus Entry Using Recombinant Virus Assays

Abstract

The present invention provides a method for identifying whether a compound inhibits entry of a virus into a cell which comprises: (a) obtaining nucleic acid encoding a viral envelope protein from a patient infected by the virus; (b) co-transfecting into a first cell (i) the nucleic acid of step (a), and (ii) a viral expression vector which lacks a nucleic acid encoding an envelope protein, and which comprises an indicator nucleic acid which produces a detectable signal, such that the first cell produces viral particles comprising the envelope protein encoded by the nucleic acid obtained from the patient; (c) contacting the viral particles produced in step (b) with a second cell in the presence of the compound, wherein the second cell expresses a cell surface receptor to which the virus binds; (d) measuring the amount of signal produced by the second cell in order to determine the infectivity of the viral particles; and (e) comparing the amount of signal measured in step (d) with the amount of signal produced in the absence of the compound, wherein a reduced amount of signal measured in the presence of the compound indicates that the compound inhibits entry of the virus into the second cell.

What is claimed is: 
     
         1 - 16 . (canceled) 
     
     
         17 . A method for determining whether one or more viral species from a population of Human Immunodeficiency Virus-1 (HIV-1) present in a sample from an HIV-1-infected patient is susceptible to an entry inhibitor comprising:
 (a) simultaneously, and in a single reaction vessel, amplifying DNA molecules comprising a portion of the HIV-1 envelope gene of the population of HIV-1 present in the HIV-1-infected patient sample to obtain a plurality of DNA molecules, each encoding the portion of the HIV-1 envelope protein from the population of HIV-1 present in the HIV-1-infected patient sample;   (b) simultaneously, and in a single reaction vessel, inserting the plurality of DNA molecules from step (a) into a first expression vector engineered to express an HIV-1 envelope protein in cells resulting in production of a plurality of distinct expression vectors, each member of which comprises only one of the plurality of DNA molecules, wherein the plurality of distinct expression vectors express a plurality of variants of the portion of the HIV-1 envelope protein that is representative of the HIV-1 species found in the HIV-1-infected patient sample;   (c) transfecting into a plurality of first cells (i) the plurality of expression vectors from step (b) and (ii) a viral expression vector which lacks the nucleic acid encoding the portion of the HIV-1 envelope protein from step (a) but includes nucleic acid encoding other proteins required for replication of the HIV-1, such that the plurality of first cells produce viral particles comprising the HIV-1 envelope proteins having sequences representative of the HIV-1 species present in the HIV-1-infected patient sample;   (d) contacting a first portion of the viral particles produced in step (c) with an entry inhibitor and a first plurality of second cells, wherein the second cells each expresses a cell surface receptor and at least one co-receptor to which the HIV-1 binds for entry, and wherein either the second cells or the viral particles comprise an indicator nucleic acid molecule which produces a detectable signal following infection of the second cells by the viral particles of step (c);   (e) contacting a second portion of the viral particles of step (c) with a second plurality of the second cells in the absence of the entry inhibitor;   (f) measuring the amount of the detectable signal produced by the first and second plurality of second cells from step (d) and step (e) in order to determine the infectivity of the viral particles in the presence and the absence of the entry inhibitor, respectively; and   (g) comparing the amount of signal measured in step (f), wherein a reduced amount of signal measured for the viral particles of step (d) as compared to the signal measured for the viral particles of step (e) indicates that at least one species from the population of HIV-1 from the HIV-1-infected patient sample is susceptible to the entry inhibitor.   
     
     
         18 . The method of  claim 17 , wherein the portion of the HIV-1 viral envelope polypeptide from the patient sample includes at least a portion of the intracellular cytoplasmic tail (CT) domain. 
     
     
         19 . The method of  claim 17 , wherein the portion of the HIV-1 viral envelope polypeptide from the patient sample does not include the intracellular cytoplasmic tail (CT) domain or a portion thereof. 
     
     
         20 . The method of  claim 19 , wherein the first expression vector includes a cytoplasmic tail domain from a genetically characterized HIV-1 virus. 
     
     
         21 . The method of  claim 17 , wherein step (d) is performed by contacting the viral particles with the entry inhibitor prior to contact with the first plurality of second cells. 
     
     
         22 . The method of  claim 17 , wherein the inhibitor binds or interferes with the binding of the HIV-1 envelope protein. 
     
     
         23 . The method of  claim 17 , wherein the inhibitor is an antibody, small molecule, or peptide. 
     
     
         24 . The method of  claim 23 , wherein the antibody binds to CD4, CXCR4, or CCR5. 
     
     
         25 . The method of  claim 23 , wherein the antibody binds to the HIV-1 envelope protein. 
     
     
         26 . The method of  claim 25 , wherein the antibody binds to the CD4 binding site of the HIV-1 envelope protein. 
     
     
         27 . The method of  claim 25 , wherein the antibody binds to the V1/V2 region or the V3 region of the HIV-1 envelope protein. 
     
     
         28 . The method of  claim 25 , wherein the antibody recognizes a specific glycosylation pattern of the HIV-1 envelope protein. 
     
     
         29 . The method of  claim 25 , wherein the antibody binds to the MPER site of the HIV-1 envelope protein. 
     
     
         30 . The method of  claim 25 , wherein the antibody binds to the CXCR4 binding site or the CCR5 binding site of the HIV-1 envelope protein. 
     
     
         31 . The method of  claim 17 , wherein the indicator nucleic acid molecule comprises a luciferase gene. 
     
     
         32 . The method of  claim 17 , wherein the receptor comprises at least one of CD4, and the co-receptor comprises at least one of CXCR4 or CCR5. 
     
     
         33 . The method of  claim 17 , wherein the plurality of DNA molecules encoding the portion of the HIV-1 envelope protein encode an HIV-1 gp160 envelope polyprotein or an HIV-1 gp120 viral surface envelope protein and at least a portion of an HIV-1 gp41 transmembrane envelope protein. 
     
     
         34 . The method of  claim 17 , wherein the viral expression vector comprises an HIV-1 gag-pol gene. 
     
     
         35 . The method of  claim 17 , wherein the first and/or second cells are human cells. 
     
     
         36 . The method of  claim 35 , wherein the human cells are human embryonic kidney cells, T cells, peripheral blood mononuclear cells, astroglioma cells, or osteosarcoma cells. 
     
     
         37 . The method of  claim 36 , wherein the human embryonic kidney cells are 293 cells, the astroglioma cells are U87 cells, and the osteosarcoma cells are HT4 cells.