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

To assess the mode of action of PT-1, we first conducted time-of-addition assays to thoroughly investigate possible targets of the HIV-1 life-cycle. The HIV-1 reverse transcriptase inhibitor AZT, fusion inhibitor T20, integrase inhibitor raltegravir, and CCR5 inhibitor maraviroc (MVC) served as controls. PT-1 was added to TZM-bl cells at different time points (pre viral adsorption 0 h, virus-cell fusion 0.25, 0.5, and 1 h, reverse transcription 2 h, integration 6 h, transcription 9 h and maturation 24 h) [34] . Cells were collected 48 h post-infection and analyzed for luciferase activity. As illustrated in Figure 2A , the reverse transcriptase inhibitor AZT exhibited potent anti-HIV activity for up to 2 h, whereas RAL began to lose inhibition activity after 8 h. PT-1, CCR5 inhibitor MVC and fusion inhibitor T20 showed inhibition from 0 h to 2 h, indicating that PT-1 inhibited a very early step of virus replication, possibly the viral entry process. We next analyzed the effects of PT-1 on the attachment of HIV-1 Env protein to target cells by comparing Env pseudotyped and VSV-G pseudotyped virus infection using TZM-bl assays. As noted in Figure 2B , inhibition was abolished after replacement of the HIV-1 envelope with VSV-G, although no differences of inhibition were observed in AZT treated cells ( Figure 2C ), indicating that PT-1 specifically interacted with the HIV-1 glycoproteins. virus replication, possibly the viral entry process. We next analyzed the effects of PT-1 on the attachment of HIV-1 Env protein to target cells by comparing Env pseudotyped and VSV-G pseudotyped virus infection using TZM-bl assays. As noted in Figure 2B , inhibition was abolished after replacement of the HIV-1 envelope with VSV-G, although no differences of inhibition were observed in AZT treated cells ( Figure 2C ), indicating that PT-1 specifically interacted with the HIV-1 glycoproteins. and AZT (C) on the replication of Env and VSV-G pseudovirions. TZM-bl cells were infected with 1000 TCID 50 (50% tissue culture infective dose)/mL Env (pREJO4541.67) and VSV-G pseudovirions, respectively. After 48 h of treatment with PT-1 or AZT, inhibition was analyzed by luciferase activity assays. All data were performed in triplicate and repeated in three independent experiments.