Human immunodeficiency virus type 1 (HIV-1) Gag protein is the principal structural component of the HIV particle. Localization of the Pr55(Gag) protein to the plasma membrane initiates virus assembly. Recent studies indicated that d-myo-phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) regulates Pr55(Gag) localization and assembly. We determined the binding affinity between Pr55(Gag) or its N-terminal MA domain and various phosphoinositide derivatives using a highly sensitive surface plasmon resonance (SPR) sensor and biotinylated inositol phosphate. The equilibrium dissociation constants obtained using this approach reflected the distinct magnitude of acyl group-based and phosphate group-based interactions. The dissociation constant (K(D)) for Pr55(Gag) complexed with 1,4,5-IP3 (an inositol with divalent phosphate groups and devoid of lipid groups) was 2170 microM, while the K(D) for di-C(8)-PI (a lipid-containing inositol devoid of divalent phosphate groups) was 186 microM, and the K(D) for di-C(8)-PI(4,5)P2 (an inositol with both lipid and divalent phosphate groups) was 47.4 microM. The same trend in affinity was observed when these phosphoinositides were complexed with MA. Our results suggest that the contribution of hydrophobic acyl chains is greater than negatively charged inositol phosphates in Pr55(Gag)/MA binding. Furthermore, each inositol phosphate (devoid of lipid groups) tested showed a distinct Pr55(Gag)-binding affinity depending on the position and number of phosphate groups. However, the position and number of phosphate groups had no effect on MA-binding affinity.

Despite the development of antiretroviral therapy against HIV, eradication of the virus from the body, as a means to a cure, remains in progress. A “kick and kill” strategy proposes “kick” of the latent HIV to an active HIV to eventually be “killed”. Latency-reverting agents that can perform the “kick” function are under development and have shown promise. Management of the infected cells not to produce virions after the “kick” step is important to this strategy. Here we show that a newly synthesized compound, L-HIPPO, captures the HIV-1 protein Pr55Gag and intercepts its function to translocate the virus from the cytoplasm to the plasma membrane leading to virion budding. The infecting virus thus “locked-in” subsequently induces apoptosis of the host cells. This “lock-in and apoptosis” approach performed by our novel compound in HIV-infected cells provides a means to bridge the gap between the “kick” and “kill” steps of this eradication strategy. By building upon previous progress in latency reverting agents, our compound appears to provide a promising step toward the goal of HIV eradication from the body.

No abstract

The precursor of Gag protein (Pr55(Gag)) of human immunodeficiency virus, the principal structural component required for virus assembly, is known to bind d-myo-phosphatidylinositol 4,5-bisphosphate (PIP2). The N-terminus of Pr55(Gag), the MA domain, plays a critical role in the binding of Pr55(Gag) to the plasma membrane. Herein, we designed and synthesized myo-phosphatidylinositol 2,3,4,5,6-pentakisphosphate (PIP5) derivatives comprising highly phosphorylated inositol and variously modified diacylglycerol to examine the MA-binding properties. The inositol moiety was synthesized starting with myo-inositol and assembled with a hydrophobic glycerol moiety through a phosphate linkage. The Kd value for MA-binding of the PIP5 derivative 2 (Kd = 0.25 μM) was the lowest (i.e., highest affinity) of all derivatives, i.e., 70-fold lower than the Kd for the PIP2 derivative 1 (Kd = 16.9 μM) and 100-fold lower than the Kd for IP6 (Kd = 25.7 μM), suggesting the possibility that the PIP5 derivative blocks Pr55(Gag) membrane binding by competing with PIP2 in MA-binding.