Jenkinson Stephen, McCoy David C, Kerner Sandy A, Ferris Robert G, Lawrence Wendell K, Clay William C, Condreay J Patrick, Smith Chari D
GlaxoSmithKline Research and Development, Research Triangle Park, NC.
J Biomol Screen. 2003 Aug;8(4):463-70. doi: 10.1177/1087057103255747.
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.
M嗜性HIV毒株进入细胞的初始事件是通过病毒包膜蛋白gp120与宿主细胞共受体CCR5和CD4相互作用。抑制这一事件可在体外减少病毒融合及进入细胞。作者利用杆状病毒介导的基因转导技术开发了一种细胞/细胞融合检测方法，该方法模拟HIV病毒/细胞融合过程，并可对这一融合事件进行高通量定量分析。该检测方法设计使用稳定转染表达CCR5、CD4和长末端重复序列（LTR）-荧光素酶的cDNA的人骨肉瘤（HOS）细胞作为受体宿主细胞。用杆状病毒构建体转导以表达病毒蛋白gp120、gp41、tat和rev的HEK-293细胞系代表病毒。gp120与CCR5/CD4相互作用导致两种细胞融合，并将tat转移至HOS细胞胞质溶胶；tat继而与荧光素酶报告基因上的LTR区域结合并激活转录，导致细胞荧光素酶活性增加。总之，已证明所开发的细胞/细胞融合检测方法是一种可靠且可重复的高通量替代检测方法，可用于评估化合物对gp120/CCR5/CD4介导的病毒融合进入宿主细胞的影响。
