Methods of evaluating cell surface receptor binding of a patient derived population of viral envelope protein constructs
View Patent ↗The 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.
1. A method for determining whether a population of viruses infecting a patient binds a cell surface receptor when entering a cell, comprising:
(a) contacting a plurality of viral particles with cells that express a cell surface receptor, wherein the plurality of viral particles comprise (i) a viral expression vector that lacks a nucleic acid encoding a functional viral envelope gene and which comprises an indicator nucleic acid that produces a detectable signal, and (ii) a plurality of viral envelope proteins, wherein the viral envelope proteins are expressed by nucleic acid molecules amplified from a sample from the patient; and
(b) detecting the detectable signal produced by the cells,
wherein detection of the detectable signal indicates that the population of viruses binds the cell surface receptor.
2. The method of claim 1 , wherein the viral particles are produced by co-transfecting into a cell (i) a plurality of nucleic acids obtained from the patient, wherein the plurality of nucleic acids encode envelope proteins from the viral population infecting the patient and (ii) a viral expression vector lacking a nucleic acid encoding a functional viral envelope protein, wherein the viral expression vector comprises an indicator nucleic acid that produces a detectable signal.
3. The method of claim 1 , wherein the indicator nucleic acid comprises an indicator gene.
4. The method of claim 3 , wherein the indicator gene is a luciferase gene.
5. The method of claim 1 , wherein the cell surface receptor is CD4.
6. The method of claim 5 , wherein the cells also express a chemokine receptor.
7. The method of claim 6 , wherein the chemokine receptor is CXCR4 or CCR5.
8. The method of claim 1 , wherein the cell surface receptor is a chemokine receptor.
9. The method of claim 8 , wherein the cells also express CD4.
10. The method of claim 8 , wherein the chemokine receptor is CXCR4 or CCR5.
11. The method of claim 1 , wherein the patient is infected with HIV.
12. The method of claim 1 , wherein the nucleic acids amplified from the sample from the patient comprise nucleic acids encoding gp120 or gp41.
13. The method of claim 1 , wherein the nucleic acids amplified from the sample from the patient comprise nucleic acids encoding gp160.
14. The method of claim 1 , wherein the viral expression vector comprises an HIV nucleic acid.
15. The method of claim 1 , wherein the viral expression vector comprises an HIV gag-pol gene.
16. The method of claim 1 , wherein the viral expression vector comprises a nucleic acid encoding vif, vpr, tat, rev, vpu, and nef.
17. The method of claim 1 , wherein the cells are mammalian cells.
18. The method of claim 1 , wherein the cells are human cells.
19. The method of claim 1 , wherein the cells are human embryonic kidney cells, human T cells, human T leukemia cells, peripheral blood mononuclear cells, astroglioma cells, or human osteosarcoma cells.
20. The method of claim 1 , wherein the cells are 293 cells, U87 cells, HT4 cells, or U37 cells.