Acquired immunodeficiency syndrome (AIDS) is one of the greatest health challenges to humans. The following research was aimed at dissecting the structural features of two key proteins in the HIV life cycle, integrase (IN) and Gag, as antiviral targets in AIDS therapy.
A novel experimental strategy was established, by combining disulfide meditated IN-DNA cross-linking with mass spectrometric foot-printing analysis, for detailed structural analysis of IN-viral DNA interactions. Full length integrase was domain-selectively linked to the respective position on viral DNA substrate. For the first time, N-terminal residue Lys 14 was found be important for viral DNA binding. In addition, we also observed a conformational change involving the connection between the core and C-terminal domains induced by DNA. These findings enable us to identify the viral DNA binding channel in the active full length IN multimer.
We further applied IN-DNA disulfide cross-linking as an in vitro assay to screen potential IN inhibitors binding at the IN catalytic site. We observed a striking difference between two chicoric acid analogs. Acetylated-chicoric acid showed concentration-dependent inhibition on IN-DNA cross-linking, while methyl N,O-bis(3,4-diacetoxycinnamoyl) serinate (MDACS) had no effect on it. Affinity acetylation and mass spectrometry was further employed to better understand the structural features of IN inhibitors and determine binding modes. Our results provide structural data for the rational design of future generation of inhibitors with potential clinical relevance.
HIV-1 Gag was investigated as a potential target for antiviral therapy. We employed mass spectrometric foot-printing to elucidate structural foundations of HIV-1 Gag interactions with its assembly cofactors phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2] and RNA. Two lysines from the matrix domain, K30 and K32, were protected in the Gag: PI(4,5)P2 complex. In contrast, nucleic acid showed strong protection of lysines K391 and K424, located at the nucleocapsid domain. In addition, a specific protection of K314, located in the capsid domain, was observed only in the presence of both PI(4,5)P2 and nucleic acid. These studies suggested that binding of both cofactors promotes protein-protein interactions involving capsid domain, which must be involved in virus particle assembly.