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 surface plasmon resonance-based competition assay was performed by immobilizing gp120 recombinant protein to react with recombinant soluble CD4 protein in solution. We noted that pre-incubation of CD4 with PT-1 caused a substantial reduction in the binding signal, whereas PT-1 did not bind directly to gp120 itself, suggesting that PT-1 competed with gp120 for binding to CD4 ( Figure 3A ). We then analyzed the binding of PT-1 to CD4 recombinant protein using a direct binding assay, where CD4 protein was immobilized on a CM5 chip, and PT-1 was run in a serial of dilutions. The average K D value for the PT-1-CD4 interaction was 1.58 µM using a 1:1 langmuir binding model ( Figure 3B ). To further elucidate the binding ability of PT-1 to the natural CD4 receptor, we co-incubated PT-1 with MT4 cells and stained the cells with anti-CD4 RPA-T4 antibodies, which bind to the D1 domain (gp120 binding domain) of CD4. FACS analysis revealed that PT-1 suppressed the fluorescence intensity in a concentration-dependent manner ( Figure 3C) ., In addition, PT-1 incubation did not induce IL-2 secretion ( Figure S3 ), suggesting that PT-1 may interfere with the gp120 binding site on the CD4 receptor without activating CD4 T cells. We also compared the effects of PT-1 on CCR5-(YU2) and CXCR4-(NL4-3) utilizing strains; however, no differences were observed. PT-1 also did not inhibit the binding of specific anti-CXCR4 and anti-CCR5 monoclonal antibodies to MT4 cells (data now shown), suggesting that PT-1 did not interact with any of the co-receptors. In addition, PT-1 incubation did not induce IL-2 secretion ( Figure S3 ), suggesting that PT-1 may interfere with the gp120 binding site on the CD4 receptor without activating CD4 T cells. We also compared the effects of PT-1 on CCR5-(YU2) and CXCR4-(NL4-3) utilizing strains; however, no differences were observed. PT-1 also did not inhibit the binding of specific anti-CXCR4 and anti-CCR5 monoclonal antibodies to MT4 cells (data now shown), suggesting that PT-1 did not interact with any of the co-receptors. We also performed native polyacrylamide gel electrophoresis (N-PAGE) to detect the effects of PT-1 on gp41 6-HB formation, as shown by N-PAGE ( Figure 4B ,C). N36 alone showed no bands because of the net positive charge, whereas C34 alone showed a clear band at a low position in the gel (Lane 1). When N36 was mixed with C34, a new band corresponding to the 6-HB formed by N36 and C34 appeared at the higher position (Lane 2). After incubation with PT-1, the density of the 6-HB band was weakened, and recovery of the C34 band was observed (Lanes 4-6). In addition, AZT did not reduce 6-HB formation (Lane 3). These data indicated that PT-1 affected the functional conformation of 6-HB. Similarly, PT-1 showed clear concentration-dependent binding affinity with the N36 peptide in SPR assays, with PT-1 bound to immobilized N36 showing an average K D of 42.8 nM ( Figure 4A ). Taken together, these data revealed that PT-1 inhibited HIV-1 Env-mediated entry by blocking the gp120 binding site of the CD4 receptor and interacting with the viral gp41 NHR. and C34 appeared at the higher position (Lane 2). After incubation with PT-1, the density of the 6-HB band was weakened, and recovery of the C34 band was observed (Lanes 4-6). In addition, AZT did not reduce 6-HB formation (Lane 3). These data indicated that PT-1 affected the functional conformation of 6-HB. Similarly, PT-1 showed clear concentration-dependent binding affi