Development of a novel high-throughput surrogate assay to measure HIV envelope/CCR5/CD4-mediated viral/cell fusion using BacMam baculovirus technology.
The initial event by which M-tropic HIV strains gain access to cells is via interaction of the viral envelope protein gp120 with the host cell CCR5 coreceptor and CD4. Inhibition of this event reduces viral fusion and entry into cells in vitro. The authors have employed BacMam baculovirus-mediated gene transduction to develop a cell/cell fusion assay that mimics the HIV viral/cell fusion process and allows high-throughput quantification of this fusion event. The assay design uses human osteosarcoma (HOS) cells stably transfected with cDNAs expressing CCR5, CD4, and long terminal repeat (LTR)-luciferase as the recipient host cell. An HEK-293 cell line transduced with BacMam viral constructs to express the viral proteins gp120, gp41, tat, and rev represents the virus. Interaction of gp120 with CCR5/CD4 results in the fusion of the 2 cells and transfer of tat to the HOS cell cytosol; tat, in turn, binds to the LTR region on the luciferase reporter and activates transcription, resulting in an increase in cellular luciferase activity. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a robust and reproducible high-throughput surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4-mediated viral fusion into host cells.
GlaxoSmithKline Research and Development, Research Triangle Park, NC.
Stephen
S
Jenkinson
David C
DC
McCoy
Sandy A
SA
Kerner
Robert G
RG
Ferris
Wendell K
WK
Lawrence
William C
WC
Clay
J Patrick
JP
Condreay
Chari D
CD
Smith
D000577:Amides / Q000494:pharmacology; D016367:Baculoviridae / Q000235:genetics*; D020148:Butyric Acid / Q000494:pharmacology; D065100:CCR5 Receptor Antagonists; D015704:CD4 Antigens / Q000378:metabolism*; D002459:Cell Fusion; D002460:Cell Line; D045744:Cell Line, Tumor; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015686:Gene Products, env / Q000378:metabolism; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism; D006678:HIV; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D006801:Humans; D010091:Oximes; D010880:Piperidines; D010957:Plasmids; D011725:Pyridines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D019713:Receptors, CCR5 / Q000378:metabolism*; D014161:Transduction, Genetic; D014162:Transfection; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
D000577:Amides; D065100:CCR5 Receptor Antagonists; D015704:CD4 Antigens; D003497:Cyclic N-Oxides; D015686:Gene Products, env; D015694:Gene Products, rev; D015696:Gene Products, tat; D015699:HIV Envelope Protein gp120; D010091:Oximes; D010880:Piperidines; D011725:Pyridines; D000644:Quaternary Ammonium Compounds; D019713:Receptors, CCR5; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D020148:Butyric Acid; C438583:Ancriviroc; C119369:TAK 779; D004121:Dimethyl Sulfoxide