The continual emergence of HIV strains that are resistant to currently approved anti-HIV drugs is an increasing threat to the effective treatment of HIV infection and control of the HIV/AIDS epidemic. Therefore, the discovery and development of new anti-HIV drugs with novel antiviral mechanisms and targets are urgently needed. The long-term objective of this application is to develop a novel class of anti-HIV drugs representing novel chemical entities targeting late stages of the HIV-1 replication cycle. Our recent studies demonstrate a novel protein- protein interaction between HIV-1 Gag and host filamin A, which is involved in late stages of the HIV-1 replication cycle in a productive manner. Disruption of the interaction redistributes Gag subcellular localization and inhibits particle release. These data suggest that the Gag-filamin A interaction could be developed as targets for HIV therapeutics. In this application, we hypothesize that small synthetic peptides, containing the binding site required for the interaction, might block specifically the interaction resulting in the impaired virus assembly and release. Experiments designed in this application will be performed to test this central hypothesis.
In specific aim 1, the binding site for Gag and filamin A will be defined by mutagenesis, as well as in vitro and in vivo binding studies. Surface plasmon resonance (SPR) will be used to evaluate the binding kinetics of the interaction. Experiments in specific aim 2 will be designed to identify peptide candidates to specifically block the interaction. Gag- and filamin A-based libraries with overlapping peptide sequences covering the binding site and its surrounding region will be generated. Screen for peptide candidates against libraries will be performed using direct binding and competitive binding inhibition assays. The binding dynamics of peptide candidates with target proteins will be characterized.
In specific aim 3, the role of peptide candidates on virus assembly and release will be examined. Cellular uptake and targeting validation of peptide candidates, which are mediated by Tat peptide, will be evaluated by fluorescence microscopy and flow cytometry. The role of peptide candidates on HIV-1 assembly and release will be determined in human T cell lines, and primary human CD4+ T cells and macrophages. Taken together, these studies will not only provide new sight into retrovirus-host interaction, but also impact the HIV/AIDS therapy by developing novel peptide inhibitors targeting the Gag-filamin A interaction.
The discovery of novel anti-retroviral drugs with new mechanisms is a high research priority due to the rapid emergence of isolates resistant to currently approved therapeutics. The information gained from our studies to identify and characterize peptide candidates targeting late stages of the HIV-1 replication cycle will impact HIV therapeutics. Our studies will take important steps toward developing the prototype of a novel class of anti-HIV inhibitors.
|Dotson, Dominique; Woodruff, Elvin A; Villalta, Fernando et al. (2016) Filamin A Is Involved in HIV-1 Vpu-mediated Evasion of Host Restriction by Modulating Tetherin Expression. J Biol Chem 291:4236-46|
|Sutton, Jessica; Dotson, Dominique; Dong, Xinhong (2013) Molecular Events in Late Stages of HIV-1 Replication. JSM Microbiol 1:|
|Liu, Ling; Sutton, Jessica; Woodruff, Elvin et al. (2012) Defective HIV-1 particle assembly in AP-3-deficient cells derived from patients with Hermansky-Pudlak syndrome type 2. J Virol 86:11242-53|
|Cooper, JoAnn; Liu, Ling; Woodruff, Elvin A et al. (2011) Filamin A protein interacts with human immunodeficiency virus type 1 Gag protein and contributes to productive particle assembly. J Biol Chem 286:28498-510|