Heldsinger A A, Antonucci T
Department of Infectious Diseases, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105.
J Virol Methods. 1994 Oct;49(3):247-55. doi: 10.1016/0166-0934(94)90140-6.
An in-vitro assay was developed to screen for HIV-1 protease inhibitors using a non-infectious proviral clone (X19) with a deletion in the envelope gene (Ratner et al., 1991). The proviral clone, X19, was expressed transiently in the COS 7 cell line. The virus was able to replicate as assessed by the presence of p24 in the supernatants, yet the virions produced did not infect CD4 positive cells. To determine the effect of a known protease inhibitor on p24 antigen production, PD 148310 (a Ro 31-8959 analog) was added immediately after transfection of the COS 7 cells. Virus particles were produced maximally after 24 h and cell supernatants were assayed for p24 antigen production using a p24 ELISA assay. PD 148310 inhibited p24 release in a dose-dependent manner with an IC50 of 23.6 nM. Western blot analysis of the supernatants using a mouse monoclonal antibody against p24 confirmed the presence of a well-defined p24 band in the control lane. At 1000 nM of PD 148310 the p24 band was not detectable, leaving only the unprocessed p55 Gag precursor. This technique is a useful tool to screen for potential HIV-1 protease inhibitors.
利用一个包膜基因缺失的非感染性原病毒克隆（X19）（Ratner等人，1991年）开发了一种体外试验，用于筛选HIV-1蛋白酶抑制剂。原病毒克隆X19在COS 7细胞系中瞬时表达。通过上清液中p24的存在评估，该病毒能够复制，但产生的病毒颗粒不感染CD4阳性细胞。为了确定一种已知蛋白酶抑制剂对p24抗原产生的影响，在COS 7细胞转染后立即加入PD 148310（Ro 31-8959类似物）。24小时后病毒颗粒产生达到最大值，使用p24 ELISA试验检测细胞上清液中p24抗原的产生。PD 148310以剂量依赖方式抑制p24释放，IC50为23.6 nM。使用抗p24小鼠单克隆抗体对上清液进行蛋白质免疫印迹分析，证实对照泳道中存在一条清晰的p24条带。在1000 nM的PD 148310浓度下，检测不到p24条带，仅留下未加工的p55 Gag前体。该技术是筛选潜在HIV-1蛋白酶抑制剂的有用工具。
