Inhibition of Human Immunodeficiency Virus Type 1 Entry by a Keggin Polyoxometalate

Wang, Xiaoli; Wang, Jiao; Zhang, Wenmei; Li, Boye; Zhu, Ying; Hu, Qin; Yang, Yishu; Zhang, Xiaoguang; Yan, Hong; Zeng, Yi

The inhibitory effect of PT-1 on HIV-1 integrase 3 processing activity was performed using the fluorescence resonance energy transfer assay as previously described [33] . The Sso7d fusion integrase was kindly gifted from Liu Wei from Beijing University of Technology. Oligonucleotide substrates were synthesized from SBS Genetech (Beijing, China). The sequences were: 5 [FAM]-ACTGCTAGAGATTTTCCACGTGGAAAATCTCTAGCAGT-[DABCYL]-3 . The DNA substrates were dissolved in the annealing buffer (10 mM, Tris-HCl, 50 mM NaCl, 1 mM EDTA, Ph = 8.0), denatured for 2 min at 94 • C, and annealed for 2 h by dropping 1 • C every 90 s. The 3 processing reaction was performed in a black solid-bottom 96-well plate. Briefly, PT-1 was incubated with 2.5 µg integrase in reaction buffer (0.25 M DTT, 200 mM NaCl, 50% glycerol, 40 mM Hepes) for 30 min at 37 • C. Subsequently, 20 pM substrates and 10 mM MnCl 2 were added to start reaction. After 4 h incubation, the fluorescence intensity was measured at Ex/Em = 485 nm/528 nm by Enspire ® Multi-mode Plate Reader. RAL was used as a positive control.